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The effects of various concentrations of cyclic AMP (cAMP) on the metabolism of androst-4-ene-3,17-dione were examined in electropermeabilized rat hepatocytes. cAMP had a biphasic effect on hepatic steroid metabolism which was dependent upon [ill] concentration and time. At low concentrations (50 µM) early (1 h) inhibitory effects predominated, whereas at higher concentrations (5 mM) later (2–3 h) stimulatory effects were seen. The use of selective protein kinase inhibitors indicated that all the effects of cAMP were mediated through activation of protein kinase A, but that the inhibitory response also involved activation of protein kinase C. The stimulatory effect was blocked by cycloheximide, indicating that protein synthesis is necessary for this response. These findings help to elucidate the mechanisms by which hormones and other compounds may give their effects on hepatic steroid metabolism and indicate a possibly novel interaction of cAMP and protein kinase C.