We have previously demonstrated that cytochrome P-450c and P-450d mRNAs can be induced by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium (HDM) plus minimal salts (Silver, G., and Krauter, K. S. (1988) J. Biol. Chem. 263; 11802-11807). Such cultures were used to investigate the role of the individual hormonal components present in the medium in the hydrocarbon-mediated induction process. Replacing HDM with minimal salts plus 10% fetal bovine serum (FBS) resulted in a 4-fold reduction in the accumulation of P-450d mRNA in response to MCA. In contrast, no effect was seen on induced levels of P-450c mRNA. Mixing experiments, in which primary cultures of hepatocytes were grown in medium containing HDM plus 10% FBS, indicated that there was no negative acting component present in FBS, but rather there was a positive acting component present in the mixture of hormones in HDM which permitted P-450d induction by MCA. Testing the effects of singly deleting each of the 10 components in HDM on MCA-induced P-450d expression demonstrated that dexamethasone was the only factor which affected the induction of P-450d. Deletion of this component from HDM resulted in a 4-fold decrease in the maximum MCA induced expression of P-450d mRNA. Moreover, supplementation of minimal salts plus 10% FBS with dexamethasone restored full P-450d inducibility by MCA. Deletion of the other components from HDM had no effects on P-450d mRNA accumulation. Although substratum clearly contributed to the quality of primary hepatocyte cultures, we were unable to demonstrate any role of the substratum on MCA induction of P-450d. In vitro nuclear run-on experiments revealed that dexamethasone had little effect on the rate of transcription of the P-450d genes. Therefore, the effect of dexamethasone on induction must be at the posttranscriptional level.