Primary cultures of human hepatocytes and hepatoma cell line HepG2 are frequently used to evaluate the hepatic disposition of drugs and other xenobiotics. To check the variability of the expression of drug-metabolizing enzymes in these in vitro models, expression of genes coding for several cytochrome P450 isoforms and phase II enzymes was quantified during culture time by real-time RT-PCR. Gene expression was determined daily for primary hepatocytes maintained in a sandwich culture over 1 week and for HepG2, during the first 10 passages. In primary hepatocytes characteristic expression trends were observed which could be abstracted into three major classes of time curves. Genes of the first and the second class had an expression maximum around day 6 and day 4 in culture, respectively. The third class of genes had two expression peaks: at day 1 and 5 in culture. Surprisingly, also the cell line HepG2 showed significant expression changes during passages. For example, gene expression of cytochrome 1A1 varied 8-fold, that of cytochrome 2B6 30-fold, and that of NADP-quinone reductase 1 more than 200-fold within the first 10 passages. In conclusion, neither primary hepatocytes nor HepG2 cell line display a model for constant expression of drug-metabolizing enzymes.