Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P-450 in animals. The expression of four cytochrome P-450 enzymes (or groups of enzymes, namely P-450 IIIA, P-450 IIC8/9/10, P-450 IIE1, and P-450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3-methylcholanthrene was ineffective. Both P-450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIIA expression was not affected by phenobarbital and was decreased by 3-methylcholanthrene. P-450s IA2 and IIE1 decreased to 25-50% of the initial level during these cultures. P-450 IA2 and ethoxyresorufin O-deethylase activity (which is a monooxygenase activity related to P-450 IA family) were increased only by 3-methylcholanthrene and did not respond to the other inducers. P-450 IIE1 was not induced by any of these compounds. P-450 IIC8/9/10 and P-450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P-450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P-450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn. These results clearly demonstrate that human hepatocytes continue to express cytochrome P-450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver.