Exposure to phenobarbital in utero results in sex-dependent altered neonatal behavior and reproductive maturation in hamsters. Prenatal phenobarbital-exposed (40 mg/kg subcutaneous to pregnant dams) male pups have at least 30% less pivoting activity and ultrasonic calling (high-frequency sounds produced by pups when separated from the nest) when compared with control pups born to saline-injected dams. As prenatal phenobarbital-treated male and female hamsters mature, there is a dose-dependent (20, 40, and 60 mg/kg) 2- to 5-day delay, respectively, according to sex, in puberty onset. Both sexes exhibited 30% lighter than control's adrenal weights associated with prenatal phenobarbital exposure. Although gonadal weights for males exposed to phenobarbital in utero were not affected, females had up to 33% lighter ovarian weights when compared with controls. Females exposed to phenobarbital in utero also had approximately 40% more irregular estrous cycles. Gonadal hormones act by means of Central Releasing Factor on pituitary ACTH secretion. Additional treatment during the neonatal period with 10 IU ACTH decreased the percentage of irregular estrous cycles and shortened the delay in puberty onset to near-control values in prenatally phenobarbital-treated females.