Behavioral sex differences have been linked to the presence of testosterone secretion during a critical perinatal period. The present experiment tested whether or not castration at different ages (early postnatal period and adulthood) would alter performance in the plus maze, a behavioral test of anxiety. Intact adult male rats (n = 17) were compared to intact adult females (n = 17); adult castrated males (n = 7) to sham-operated adult male rats (n = 9); and newborn castrated males (n = 7) to sham-operated male offspring (n = 8). When adult, the subjects were left on an elevated plus maze for 5 min. Females made a higher percentage of entries onto the open arms and showed a greater number of scans over the edge of an open arm than males. There were no differences in the percentage of arm entries or time spent on the open arms when adult castrated males were compared to sham-operated rats. On the other hand, newborn castrated males showed a significantly higher number of open arm entries and spent a greater percentage of time on the open arms than sham-operated offspring. The results demonstrate that the absence of male gonadal hormones during the perinatal period decreases anxiety, as assessed in the elevated plus maze, leading to a behavioral pattern that resembles that of females. These data provide evidence for the organizational role of gonadal hormones in the development of behavioral inhibitory systems.