Background Multiple sclerosis (MS) is a demyelinating disease of the central nervous system of unknown aetiology. The geographical distribution of MS shows that her frequency can also vary inside territorially closed areas. Objective To determine the epidemiological, clinical, serologic, immunologic and genetic differences between MS-patients and controls in the Municipalities of (high-risk area) and Vrbovsko (low-risk area), which are situated in Gorski kotar, area of high-risk for MS in Croatia. Methods and subjects Retrospective study of 19 patients from Municipality of Čabar and 9 patients from Municipality of Vrbovsko, clinically definite cases of MS (Poser's criteria), and double number of controls, has been carried out between 2001-2004. Plan of investigation included evaluation of descriptive epidemiological rates and case-control studies ("door to door" interview) about demographic data, profession, socio-economic status, specificity of personalities, personal and family anamnesis, specificities of housing, specificities of fertility period in women, breast feeding and nutrition, exposure to animal contact before age of 18. Blood sample was taken from each individual for biochemical, serologic, immunologic and genetic analysis. Main Outcome Measures Analysis was carried out for all MS patients present in Municipality of and Municipality of Vrbovsko on the prevalence day of March 31- 2001 in order to calculate the prevalence, incidence and mortality of MS, and then for each indicator, in order to calculate the Odds Ratio (OR) and 95% Confidence Interval (CI) for all indicators that were more frequent in patients then in controls, and vice versa. Results In the Municipality of the crude prevalence of MS was 160.3 per 100.000 inhabitants; annual mean incidence (1984-2001) was 5.52 per 100.000 inhabitants per year (95% CI, 3.27-8.72); annual mortality rate (1946-2001) was 2.76 per 100.000 inhabitants (95% CI, 1.26-5.24); sex ratio (F/M) was 1.11; mean age of onset was 28.53 (10.04) years; mean duration of disease to prevalence day was 27.26 (11.11) years; an affected family member was identified in 52.63% of the autochthonous population; mean diagnostic time was 4.5 (6.58) years. In the Municipality of Vrbovsko crude prevalence of MS was 66.14 per 100.000 inhabitants; annual mean incidence (1948-2001) was 1.99 per 100.000 inhabitants per year (95% CI, 0.91-3.78), (P=0.009); annual mortality rate (1946-2001) was 0.88 per 100.000 inhabitants (95% CI, 0.24-2.27), (P=0.06); sex ratio (F/M) was 1.25; mean age of onset was 27.52 (8.58) years (P=0.79); mean duration of disease to prevalence day was 19.34 (9.52) years (P=0.264 ); an affected family members was identified in 11.1 % of the autochthonous population (P=0.042); mean diagnostic time was 5.27 (4.48) years. In both municipalities, the age-specific prevalence within both genders prevailed in the 30-49 age group. In the Municipality of MS patients were all Croats, descendants of croatized Germans and/or Slovenians (P=0.026). In the Municipality of Vrbovsko MS patients were mostly Serbs (P=0.026). Variables connected to significant risk of MS in the Municipality of included professions in agriculture, farming and forestry professions (P=0.007), allergies in patients (P=0.019) and family members (P=0.0018), injuries (P=0.002), usage of salt with nitrites (P=0.03), alcohol consumption (P=0.0009), food rich in animal fats (P=0.000001); consumption of oat and it's products (P=0.0057), light colors of hair and eyes (P=0.05), open type of sewage system (P=0.000013), constant area of residence until age of 18 (P=0.017) and family history of MS (P=0.0046). Sumames of familiar MS patients were all of German (30%), Slovenian (40%) and Slovenian-German (20%) origin. Female MS patients from Municipality of were mostly housewives (P=0.029) with early menarche (P=0.018), early conception, pregnancy and birth (n.s.). No connection was found with dental fillings and structures (P=0.010), regular consumption of fruits and vegetables (P=0.009), fresh fish (P=0.002), frozen fish (P=0.00007), meat of wild animals (P=0.03), meat products dried on the air (P=0.004), usage of water from the pipe (P=0.011), closed sewage system (P=0.000013), contact with domestic animals (P=0.01) and with the house made of bricks (P=0.01). In the Municipality of Vrbovsko MS patients were eating more food rich in animal fats (P=0,03), beans (P=0,027), consumption of alcohol (P=0,006), living in wooden houses (P=0,067) and were rarely drinking unboiled water (P=0,05). Initial symptomatology of MS (mainly motor and visual symptoms), clinical course and development of disability were similar in both groups of patients. Associated diseases were present within the MS patients from Municipality of and they had more frequent complications during various therapeutic procedures. Concentration of serum antibodies was significantly increased only within group of MS patients from Municipality of Čabar as follows: against cytomegalovirus (P=0.00025), Epstein-Barr virus (P=0.006), parotitis virus (P=0.037) and respiratory syncytial virus (P=0.008). Serologic tests for Hepatitis B virus was positive in 10.5% MS patients from Municipality of Čabar (P=0.18) and they shown four times higher infestation with Toxoplasma gondii then controls. IgG antibodies to Borrelia burgdorferi were present in the blood of 2% of patients. Alleles -A3 (P=0.03) and -DR2 (P=0.004) were present only in the MS patients from Municipality of Čabar. Conclusion Our investigation confirms that genetic, ethnic, sociological, linguistic and cultural differences of neighboring zones in Gorski kotar, Croatia are responsible for heterogenic distribution of MS in this area. In the Municipality of familiar (pseudo)cluster of MS, «founder effect» is present maintaining the greater risk for MS. Allergy diathesis and mainly fat/meat dietary regime, alcoholism and low hygiene-sanitary standard amongst ecologic factors, play a role in the etiopathogenesis od MS in genetically disposed individuals in both Municipalities.