The development of physiological responsiveness to male stimuli in peripubertal female gray short-tailed opossums was examined in this study. Prepubertal females were exposed directly or indirectly to male chemicals (odors) or to no male stimuli. All females exposed directly to deposited male stimuli (22/22) showed estrus at a mean (+/- SEM) age of 127 +/- 2 days. Only 59% (13/22) of females exposed indirectly showed estrus, and at an older mean age of 162 +/- 5 days (p less than 0.01). Vaginal estrus was not observed in any of the females isolated from male stimuli (0/11). Direct exposure of prepubertal females to male odors was associated with higher uterine weights compared to those of indirectly exposed and isolated females. Reproductive success, measured as litter production, of peripubertal females greater than 140 days old was comparable to adult reproductive success. This study has shown that reproductive maturation in pubertal female opossums requires male stimuli, that this stimulus appears to be pheromonal, and that direct exposure to deposited male odors is the most effective stimulus for female reproductive maturation.