1. Rat hepatic cytochrome P450IIE1 is an ethanol-inducible enzyme which catalyses ethanol oxidation and activation of the procarcinogen, N-nitrosodimethylamine (NDMA) to its carcinogenic metabolite. 2. Initial studies in adult rat indicated that the regulation of cytochrome P450IIE1 is complex, therefore we strove to identify a central regulatory mechanism, using primary monolayer hepatocyte culture. These studies examined the effect of a range of agents (i.e. inducers, hormones, sodium butyrate and 5-aminolaevulinic acid) on amounts of cytochrome P450IIE1 protein and mRNA expression in rat hepatocytes maintained in serum-free medium on both Vitrogen and Matrigel, a laminin-rich basement membrane. 3. At time 0, immunoreactive cytochrome P450IIE1 protein was easily detectable in control cultures, yet decreased rapidly with time in culture to nearly undetectable levels at 120 h. Addition of inducers (notably, pyrazole) to the culture medium increased cytochrome P450IIE1 above that of untreated cultures at similar time points, yet did not elevate cytochrome P450IIE1 or NDMA demethylation above their levels at time 0. 4. Cytochrome P450IIE1 hybridizable mRNA also rapidly declined in culture. The decline in mRNA was not significantly altered in cultures exposed to pyrazole or any other agent. Thus, post-transcriptional factors appear to play an important role in the regulation of hepatic cytochrome P450IIE1, with protein stabilization being the most probable mechanism.