Objectives: Considering that both, the matrix metalloproteinase (MMP) and the fibrinolytic (FB) system contribute to the increasement of blood-brain barrier permeability and at the same time take part in demyelinating, inflammatory and degenerative processes in multiple sclerosis (MS) patients, the aim of our study was to analyze whether specific gene polymorphisms of MMP (MMP-9, MMP-2, TIMP-1, TIMP-2) and FB system (TPA and PAI-1), and their interactions, impact MS susceptibility and/or clinical phenotype of the disease. Patients and methods: A total of 560 MS patients and 678 control subjects were recruited from Croatian and Slovenian population, including 100 patients and 120 healthy subjects from areas with high-risk for MS (autochthonous inhabitants of Gorski kotar and Kočevje). The identification of -1562 C/T MMP-9, -1575 G/A MMP-2, -418 G/C TIMP-2, 372 C/T TIMP-1 and -675 4G/5G PAI-1 gene polymorphisms was performed by the polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) method, while for Alu I/D TPA polymorphism detection an allele-specific PCR was used. Results: Given the fact that there were no significant differences in genotype or allele distributions between MS patients and controls or when data was compared according to gender or disease course, matrix metalloproteinase gene (MMP-2, MMP-9, TIMP2, TIMP-1) polymorphisms, alone or in interaction, influenced neither MS susceptibility nor the disease course. Polymorphic variants of the FB genes (TPA and PAI-1), alone or in interaction, as well failed to show significant association with MS susceptibility or disease course in female patients. In male patients there was a significantly higher frequency (p= 0,014; OR= 1,65, CI: 1,11 - 2,46) of MS high-risk genotype combination, I+/5G+ (62,2%), than in control subjects (50,0%). Further analysis of the impact of these polymorphisms on clinical phenotype revealed the combined effect of -1562 C/T MMP-9 and -372 C/T TIMP-1 gene polymorphisms on the age of disease onset (p=0,049) in male patients with RR+SP disease course, but otherwise, there was no other significant association of MMP-2, MMP-9 and TIMP-1 gene polymorphisms or their combinations. The -1575 G/A MMP-2 polymorphism alone showed significant associations (p=0,008) in female RR+SP patients and when combined with -1562 C/T MMP-9 polymorphism showed a trend (p=0,062) for influence on EDSS. The -1562 C/T MMP-9 polymorphism was significantly associated with age of disease onset (p=0,038) but once combined with -372 C/T TIMP-1 polymorphism this association was insignificant (p=0,175). There was no significant association of TPA I/D and PAI-1 4G/5G polymorphisms with any of the regarded MS clinical characteristic in RR+SP patients of both genders. Significant association of MMP-2 polymorphisms (p=0,007) and MMP-2/MMP-9 (p=0,021) combined effect on MSSS were observed in patients with PP disease course, as well as the association of TPA I/D polymorphism with PI in male PP patients (p=0,016), and the association of PAI-1 4G/5G (p=0,028) alone and in interaction with TPA I/D (p=0,011) with age of disease onset in female patients, but due to the small number of patients in the mentioned groups this result does not allow firm conclusions. Furthermore, when comparing patient subgroups with different first MS symptoms, the distributions of MMP-2, MMP-9, TPA I/D, PAI-1 genotypes, as well as the combinations of MMP-2/MMP-9 and TPA/PAI-1 genotypes, have not differed significantly between groups with ON, MM and SM disease onset. Greater number of significantly different distributions of TIMP-1 and MMP-9/TIMP-1 genotypes in both genders indicates that the mentioned genes could really impact the type of first manifestation of the disease. Polymorphic variants of the analysed genes have as well associated with observed clinical characteristics in subgroups of patients with different first symptoms, namely the –1575 G/A MMP-2 gene polymorphism has significantly modified the age of disease onset in patients with ON (p=0,009) as a first presenting symptom, while in patients with MM there was a significant combined effect of TPA/PAI-1 (p=0,025) and MMP-9/TIMP-1 gene polymorphisms (p=0,045 in men and p=0,031 in women) on MSSS. Comparison of the polymorphisms of the MMP and FB systems in MS patients inside and outside the high- risk areas was of special importance. No significant differences were noticed regarding their influence on MS susceptibility and on the clinical phenotype of the disease, therefore the noticed differences in MS course or the clinical characteristics of the disease between the aforementioned groups cannot be attributed to the influence of the investigated polymorphisms. The analysis of the interaction between MMP and FB gene polymorphisms on the predisposition and clinical expression of the disease in our patients revealed that no single combination of MMP-9/TIMP-1 genotypes in interaction with high-risk or protective combinations of TPA/PAI-1 genotypes did not significantly influence the development or the clinical characteristics of the disease regarding age at disease onset, EDSS, MSSS, duration of the disease and PI. Conclusions: Based on the obtained results we can conclude that despite the fact that the particular polymorphic variants of the examined genes, whether alone or combined, exhibited a strong influence on the development and clinical characteristics of the disease in particular subgroups of patients stratified according to gender or MS course, in our study we have not ascertained a significant influence of the interactions of matrix metalloproteinase and fibrinolitic system gene polymorphisms on MS susceptibility and disease phenotype in Croatian and Slovenian patients.