BACKGROUND: Ascertainment of fetal sex is important in the equine industry for economic reasons. As a result, various methods have been developed for the sex determination of a fetus in a horse; however, the current techniques have some limitations. Recently, evaluation of maternal testosterone concentration has been suggested as an easy and inexpensive method for diagnosing fetal sex, but the findings are discrepant in different species. OBJECTIVES: The aim of the present study was to measure the concentration of circulating testosterone in mares carrying male and female fetuses in order to assess the diagnostic value of maternal testosterone concentration for sex determination of equine fetus. METHODS: Blood samples were collected from mares (n=20) at months three, six, and nine of pregnancy. The samples were centrifuged and stored at -20°C until hormonal analysis of testosterone concentration using an ELIZA kit. The gender of foals was determined at birth based on observation of external genitalia. RESULTS: Neither testosterone concentration in the third, sixth and ninth months of pregnancy nor the cumulative concentration of testosterone differ between mares with male and female fetuses (P>0.05). However, testosterone concentration changed during pregnancy in all mares regardless of the gender of their fetus, and it was higher at month six than at months three and nine (P<0.0001). CONCLUSIONS: In conclusion, the present study showed that maternal testosterone concentration could not be used for the sex determination of a fetus in a horse. Yet the current study revealed testosterone concentration dy-namics over various gestation stages in mares.