Primary cultures of adult rat hepatocytes grown in serum-free hormonally defined medium were shown, for the first time, to be capable of supporting the 3-methylcholanthrene-inducible expression of cytochrome P-450d. Such cultures were used to investigate the mechanism of the induction of cytochrome P-450c and P-450d mRNAs. After 1 day of growth in culture, P-450c and P-450d mRNAs were induced 33- and 28-fold, respectively, by 3-methylcholanthrene treatment. A similar magnitude of induction was achieved after 2-5 days growth in culture. However, the relative abundance of the two mRNAs before and after treatment decreased linearly over the 5-day time course. Kinetic analysis revealed that induction of both genes was rapid and could be observed less than 2 h following treatment. Accumulation of both mRNAs was linear for 8 h, reaching a plateau by 12 h. Expression then remained constant for at least 12 additional hours. In vitro nuclear run-on experiments revealed a 3.9- and 2.0-fold transcriptional induction of the P-450c and P-450d genes, respectively. This is in contrast to the large induction of accumulation of these mRNAs observed at steady state. Thus, the 3-methylcholanthrene induction of P-450c and P-450d mRNAs in the hepatocyte cultures appeared to be mediated primarily at the post-transcriptional level. Experiments on rat liver showed that, in vivo, P-450d is also regulated primarily at the post-transcriptional level. However, P-450c was found to be regulated primarily transcriptionally.