Hepatocytes cultured on matrigel express many liver-specific functions, but the levels and activities of the predominant male-specific rat hepatic CYPs, 3A2 and 2C11, decline rapidly in culture. Metyrapone maintains the level of total cytochrome P450 of rat hepatocytes in primary culture, but the mechanism underlying this effect has not been completely elucidated. The present study sought to determine whether metyrapone acts solely to stabilise CYP proteins in rat hepatocytes cultured on matrigel, or whether it also influences mRNA levels of the encoding genes. Metyrapone maintained the level of total cytochrome P450 in cultured hepatocytes so that values were > 200% of those found in untreated control cells 24 hr after isolation. At this time, CYP3A2-mediated testosterone 6 beta-hydroxylation was approximately 7-fold higher in hepatocytes cultured in the presence of metyrapone than in control cells, and CYP2C11-dependent testosterone 2 alpha- and 16 alpha-hydroxylation activities were between 2 and 3-fold greater. The results inferred from catalytic activities were supported by immunoquantitation of CYP3A and 2C11 proteins. The trend of increased CYP protein levels in metyrapone-treated cells continued throughout the 48-hr culture period. In control cells, CYP3A2 and 2C11 mRNA levels fell abruptly in culture to reach values at 24 hr that were < 30% of those in freshly isolated cells; addition of metyrapone failed to arrest this fall. However, treatment of cells with metyrapone considerably elevated levels of one or more CYP3A subfamily mRNA species, as detected by a riboprobe based on the cDNA for CYP3A1 ("CYP3A1-like mRNA') that were demonstrated, by another riboprobe, not to be CYP3A2 or RNCYP3AM. RT-PCR of mRNA prepared from cultured hepatocytes, followed by restriction mapping of the cloned cDNAs was used to characterise the CYP3A induced by metyrapone. This revealed that elevated levels of the CYP3A1-like mRNA were attributable to induction of RL33/cDEX mRNA; there were no CYP3A1 cDNAs isolated from these cells. These data are interpreted as indicating that metyrapone stabilises the expression of cytochrome P450 in culture by both pre- and posttranslational mechanisms. The particular mechanism employed is gene-specific, whereby even the highly homologous genes CYP3A2, RL33/cDEX and, possibly, RNCYP3AM are subject to different types of regulation in the presence of metyrapone.