Studies were conducted to evaluate the possible induction or the maintenance of cytochrome P-450IIE1 in primary hepatocyte cultures by the inducing agent 4-methylpyrazole. Hepatocytes were isolated from control (noninduced) rats and from rats treated in vivo with either pyrazole or 4-methylpyrazole to induce P-450IIE1. The content of P-450IIE1 was determined by Western blots with antipyrazole P-450 IgG, and catalytic activity was assessed by assays of dimethyl-nitrosamine demethylase activity. The treatment with 4-methylpyrazole in vivo increased the content of P-450IIE1 and dimethylnitrosamine demethylase activity sevenfold and fourfold, respectively. In cultures prepared from noninduced hepatocytes, P-450IIE1 levels fell to values of 76%, 65%, 31% and 1% of freshly isolated hepatocytes after 1, 3, 6 and 9 days in culture. A similar decrease in dimethylnitrosamine demethylase was observed during this time. In cultures prepared from induced hepatocytes, the decline in P-450IIE1 was more rapid as levels fell to 77%, 31%, 3% and 3% of initial values after 1, 3, 6 and 9 days in culture. Again, the fall in dimethylnitrosamine demethylase activity paralleled the decline in content of P-450IIE1 and was more rapid with the induced hepatocytes. With cultures prepared from noninduced or induced hepatocytes, the addition of 4-methylpyrazole in vitro did not increase the content of P-450IIE1 or the activity of dimethylnitrosamine demethylase over the initial values. However, 4-methylpyrazole appeared to stabilize the P-450IIE1 and to decrease its rate of decline in culture. In noninduced cultures, the percent remaining content of P-450IIE1 after 6 days was 31% in the absence of and 52% in the presence of 5 mol/L 4-methylpyrazole. In cultures from 4-methylpyrazole—induced hepatocytes, the percent remaining P-450IIE1 after 3 days was 31% in the absence of inducer and 59% with 4-methylpyrazole added in vitro. Similarly 4-methylpyrazole helped to prevent the rapid decline of dimethylnitrosamine demethylase activity in induced and noninduced cultures. Viability of the induced and noninduced cultures in the absence or presence of added 4-methylpyrazole was similar. Levels of mRNA for P-450IIE1 were similar for livers from control rats and from rats treated in vivo with 4-methylpyrazole. The mRNA levels rapidly declined in induced and noninduced cultures, and this decline, unlike the fall in P-450IIE1 or dimethylnitrosamine demethylase activity, could not be prevented by the addition of 4-methylpyrazole in vitro to the cultures. These results suggest that the already induced P-450IIE1 isozyme is more labile and subject to rapid decline in culture and that inducers such as 4-methylpyrazole appear to stabilize the P-450IIE1 and thus help to maintain this isozyme and associated catalytic activity in cultures prepared from noninduced and induced hepatocytes. (HEPATOLOGY 1990;12:1379–1389).