Men and women differ in temperament and personality traits, such as aggression and sensation seeking. The sex hormone testosterone could play a role in the origin of these differences, but it remains unclear how and when testosterone could have these effects. One way to investigate the prenatal exposure effect of testosterone is to compare opposite-sex (OS) and same-sex (SS) female twins. It has been suggested that OS twin girls are exposed prenatally to elevated testosterone levels and that this may result in some masculinization of their personality and behavior. We measured sexually dimorphic traits and circulating testosterone levels in 13-year-old OS (n = 74) and SS (n = 55) twins. Testosterone levels showed a clear circadian rhythm, with higher levels in the morning than in the afternoon. Testosterone was higher in boys than girls, but similar in OS and SS twin girls. Testosterone was not in any way systematically related to the different personality traits. However, a sex difference in aggression proneness was observed, and OS girls showed a more masculine pattern of aggression proneness than the SS girls. It is argued that it is unlikely that this difference is due to social factors, such as a gender-specific upbringing.