We used primary nonproliferating cultures of adult rat hepatocytes to investigate the regulation of P-450c and P-450d, immunochemically related protein products of separate cytochromes P-450 genes that are coinduced by 3-methylcholanthrene and related compounds. In cultures of hepatocytes prepared from untreated rats and incubated in media containing 3-methylcholanthrene, beta-naphthoflavone, 3,4,3',4'-tetrachlorobiphenyl, and Aroclor 1254 (a mixture of chlorinated biphenyls) there was a 5- to 15-fold accumulation of P-450c protein (quantitated by immunoblotting), accompanied by an increased rate of P-450c synthesis (measured as incorporation of [3H]leucine into immunoprecipitable protein) and an increased amount of P-450c mRNA hybridizable to a specific cloned cDNA (p210). In contrast, there were no increases in the concentration of P-450d protein, its rate of synthesis, or the amount of P-450d mRNA hybridizable to its specific cDNA (p72). Similarly, when "preinduced" hepatocytes (isolated from rats treated with Aroclor 1254) were incubated for 4 days in culture medium, the amount of P-450c, its rate of synthesis, and the amount of P-450c mRNA remained elevated, whereas synthesis of P-450d and the amount of P-450d mRNA fell precipitously to less than 10% of the initial values despite the presence or absence of Aroclor 1254 or of isosafrole in the medium. However, the loss of P-450d protein in these cultures was almost completely prevented when isosafrole was added to the culture medium and was partially prevented when safrole, Aroclor 1254, and 3,4,5,2',4',5'-hexachlorobiphenyl, but not 3-methylcholanthrene, beta-naphthoflavone, or 3,4,3'4'-tetrachlorobiphenyl, were in the culture medium. Moreover, in similar cultures of "preinduced" hepatocytes that were pulse-labeled with [3H]leucine, the presence of isosafrole in the culture medium extended the apparent half-life for loss of radioactivity in immunoprecipitable P-450d to a value of 72 h (3-fold longer than in standard medium) but was without effect on the rate of disappearance of radiolabeled P-450c. We conclude that control of P-450d degradation is an important factor in the regulation of this hemoprotein and that induction of P-450c and P-450d proceed by separate pathways that are spontaneously divorced under standard conditions for primary culture of adult rat hepatocytes.