Hepatic enzymes that metabolize endogenous and xenobiotic compounds have been shown to be altered in adult rats that had been exposed to xenobiotics as neonates. Protein kinase C (PKC) is important in intracellular signaling and has been implicated in the regulation of hepatic monooxygenases. Therefore, we examined the effects of neonatal exposure to diethylstilbestrol (DE5) and phenobarbital (PB) on hepatic microsomal testosterone metabolism and on the alpha form of protein kinase C (PKCα) in adult rats. In adult males, neonatal exposure to DES altered adult testosterone metabolism such that 7α‐hydroxylation was increased by 58% but 2α‐, 16α‐, and 6β‐hydroxylations and conversion to androstenedione were decreased 31–44%. In contrast, adult males neonatally exposed to PB showed increased (20–27%) testosterone 2α‐ and 16α‐hydroxylations and androstenedione formation, but no effect was observed in the rate of 6β ‐ or 7α‐hydroxylations. Western blot analyses indicated that cytosolic PKCα levels in male rats neonatally exposed to PB were decreased by ∼63% relative to the vehicle control group but were not significantly altered in the DES males. The PKCα levels generally correlated (r =‐.75) with 16α‐hydroxytestos‐terone formation in all samples. These results show that neonatal treatment with DES or PB differentially alters hepatic monooxygenase enzyme activities and PKCα levels in adult rats.