The effects of neonatally administered phenobarbital (PB) on adult rat hepatic microsomal metabolism of testosterone were examined in 60-, 90-, and 120-day-old animals. Phenobarbital-induced imprinting was evident at all ages; however, female rats appeared to be more susceptible to the neonatal effects of phenobarbital than did male rats. In 60-day-old female rats, increased testosterone 2 alpha-hydroxylase activity was observed in microsomes from noninduced rats, whereas decreased testosterone oxidation at all positions except 2 alpha and 15 beta was observed in microsomes from Aroclor 1254-induced rats. The decreased oxidation of testosterone at specific sites in response to Aroclor 1254 induction was quite dramatic, decreasing the activities to near or below control levels. By contrast, phenobarbital-treated 60-day-old males exhibited approximately a twofold increase in Aroclor 1254-induced 16 alpha and 2 alpha-hydroxylase activities. The pattern of changes in testosterone metabolism observed in phenobarbital-treated animals was different at both 90 and 120 days from that at 60 days. Only minor alterations in the oxidation of testosterone were observed in 90-day-old animals of either sex. In 120-day-old animals the greatest effects of neonatal phenobarbital exposure were on Aroclor 1254-induced 16 beta-hydroxylase activities. In induced female rats 16 beta-hydroxylase activity was again decreased to noninduced levels, while in induced male rats a fourfold increase in this activity was observed. The results demonstrate that neonatal exposure to phenobarbital can alter both constitutive and Aroclor 1254-induced testosterone metabolism in adult rats and that the effects of neonatal phenobarbital exposure are age and sex differentiated.