The influence of neonatal androgen status on the ability of neonatally administered phenobarbital (PB) to program increases in adult rat hepatic microsomal enzyme activities was investigated. Male and female rat pups were injected with 30 mg/kg PB or 0.9% saline on Days 1 to 5 postpartum. Males were further divided into either neonatally castrated or sham-operated groups. Females were subdivided into groups receiving either 0.5 mg testosterone propionate or 50 microliters corn oil on Days 1, 3, and 5 postpartum. Various hepatic microsomal cytochrome P-450 system and glucuronyltransferase activities were determined at 140 days of age. Our results indicate that PB imprints both males and females. P-450 reductase, ethoxycoumarin O-deethylase, and testosterone glucuronyltransferase activities were increased to a greater degree in females than in males. These same activities were increased by neonatal PB to a greater degree in castrated males compared to sham-operated males and were increased to a lesser degree in neonatally testosterone-treated females compared to female corn oil controls. It therefore appears that neonatal testosterone presence not only alters the patterns of enzyme activity but also diminishes the response of certain activities to PB programming.