Levels of testosterone (T) and sex hormone-binding globulin (SHBG) were measured in blood samples from pregnant women and related to gender role behavior in 342 male and 337 female offspring at the age of 3.5 years. Gender role behavior was assessed using the Pre-School Activities Inventory, a standardized measure on which a parent indicates the child's involvement with sex-typical toys, games, and activities. Levels of T, but not SHBG, related linearly to gender role behavior in preschool girls. Neither hormone related to gender role behavior in boys. Other factors, including the presence of older brothers or sisters in the home, parental adherence to traditional sex roles, the presence of a male partner in the home, and maternal education, did not relate to gender role behavior in this sample and did not account for the relation observed between T and behavior. Although other, unmeasured factors may explain the relation, the results suggest that normal variability in T levels prenatally may contribute to the development of individual differences in the gender role behavior of preschool girls.