Wound repair is a complex process that involves an integrated response by many different cell types controlled by a variety of growth factors. During the initial inflammatory phase fibroblasts enter the wound where they synthesize and remodel new extracellular matrix. Fibroblast cell cultures are often used in the design of in vitro wound model for preliminary investigations of biocompatibility and potential wound healing effect of active compounds or innovative delivery systems, through the influence on cell migration and proliferation. In this study, the biocompatibility of melatonin-loaded lecithin/chitosan nanoparticles with skin fibroblasts was investigated. Nanoparticles were prepared by ionotropic gelation of lecithin and chitosan. Four types of chitosan used differed in molecular weight (50-150 kDa or 150-400 kDa) and/or deacetylation degree (75-90 % or >90 %). Mean diameter and zeta potential of nanoparticles depended on molecular weight and deacetylation degree of chitosan and ranged between 237.8 and 263.4 nm and 25.7 and 32.1 mV, respectively. Melatonin loadings were up to 4.2%. Biocompatibility of nanoparticles with skin fibroblasts was determined by evaluation of metabolic activity of cells treated with nanoparticle suspensions at chitosan concentration ranging from 1.25 to 20 μg/ml. Apart from chitosan concentration, biocompatibility of nanoparticles was related to chitosan deacetylation degree. All types of nanoparticles induced a significant decrease in the cell viability at chitosan concentration of 10 and 20 μg/ml. Viability of cells treated with nanoparticles prepared with chitosan of higher deacetylation degree decreased significantly even at chitosan concentration of 5 μg/ml. Chitosan solutions exerted higher cytotoxic effect compared to nanoparticle suspensions. Cytotoxicity studies revealed the chitosan concentration in the system at which it is feasible to study in vitro wound healing effect of lecithin/chitosan nanoparticles, and that is 5 μg/ml for nanoparticle suspensions prepared with chitosan of lower deacetylation degree, and 2.5 μg/ml for nanoparticle suspensions prepared with chitosan of higher deacetylation degree.