Two cultured cell lines, called Kan-R1 and Kan-R2, were established from rat hepatic cells by in vitro culture with a hepatocarcinogen, 3-methoxy-4-aminoazobenzene, and examined for the gene expression of cytochrome p450 (p450) isoforms, CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP3A1 and CYP3A2, by the RT-PCR method. It was revealed that all the p450 genes examined were expressed in both cell lines, although the two cell lines differed in cell size and colony-forming ability on a soft agar. The expression levels of the CYP1A2, CYP2B1, CYP2B2, CYP3A1, and CYP3A2 genes were lower than those in liver tissues, while that of CYP1A1 was higher in the cell lines. In both cell lines, cycloheximide, an inhibitor of protein synthesis, augmented the gene expression of the p450s except CYP2B1. These findings indicate that the newly established hepatic cell lines substantially express the p450 genes for CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP3A1, and CYP3A2, and that the constitutive gene expression of these p450s, with the exception of CYP2B1, may be inhibited by negative transcription factors.