Data from lower mammals suggest a masculinizing or defeminizing influence of pre- or perinatal diethylstilbestrol (DES) exposure on various aspects of the sex-dimorphic behavior (including juvenile rough-and-tumble play) of genetic females. However, three previous studies on childhood play and adult gender-role behavior in human females have led to ambiguous results. In a follow-up study of 60 women with prenatal exposure to DES and 26 controls, we used psychometrically well-designed multi-item scales based on self-report inventories for the assessment of these aspects of behavior. No effects of DES could be demonstrated. We conclude that, at the doses studied, prenatal DES exposure in human females has not led to behavioral masculinization or defeminization of childhood play and adult gender-role behavior.