Abstract In this investigation, the genotoxic properties of mestranol were examined in vitro. Human lymphocyte cultures were exposed for 72 h to mestranol at concentrations of 7.5, 15 and 30 µg/g. The genotoxic effects of the chemosterilant were assessed by numerical and structural chromosome aberrations. Mestranol induced certain genotoxic effects in human lymphocytes. There was a dose-dependent significant (p<0.01) increase in the number of numerical aberrations in comparison to the control, but without significant differences (p>0.05) between the doses applied. Further, structural aberrations increased significantly (p<0.01) in the presence of mestranol, being most frequent in cultures exposed to the highest mestranol dose. The frequency of Robertsonian translocations increased significantly only in cultures treated with mestranol at concentration of 30 µg/g in comparison both with the control (p<0.01) and the lowest chemosterilant dose (p<0.01). There were significant differences (p<0.01) in the levels of chromosome gaps and fragments compared to Robertsonian translocations, whilst the frequencies between gaps and fragments were not significantly different (p>0.05).