The ability of phenobarbital (PB) to neonatally "imprint" or "program" the hepatic microsomal cytochrome P-450-dependent monooxygenase system (MOS) was investigated. Phenobarbital (30 mg/kg) was administered subcutaneously to neonatal rats of both sexes on days 1-5 postpartum. Various hepatic MOS activities were measured at 6, 22, 50 and 140 days of age. Six-day-old animals of both sexes displayed the increased hepatic microsomal protein levels and enzyme activities expected from the action of phenobarbital as a transitory MOS inducer. Most of these increases dissipated by 22 and 50 days of age. However, at 140 days of age rats of both sexes that had received neonatal phenobarbital showed increased levels of cytochrome P-450, as well as both P-450 and cytochrome c reductase, ethoxycoumarin O-deethylation, glucuronyl transferase activity, in vitro covalent binding of benzo[a]pyrene to DNA and in vivo covalent binding of aflatoxin B1 to hepatic macromolecular fractions. Neonatal phenobarbital administration can alter the metabolic profile of rats in adulthood, apparently by a mechanism different from that responsible for either transitory PB induction or testosterone imprinting.