We sought to establish an in vitro system to study the regulation of highly differentiated hepatocellular functions, and specifically the time-dependent expression of four cytochrome P450 (P450) genes at the messenger RNA (mRNA) and protein levels. When seeded onto matrigel, hepatocytes could be maintained for 8 days in media that were free of serum and hormones (except for insulin). Cells retained a spherical phenotype; they secreted albumin and not alpha-fetoprotein; and the cellular RNA/DNA ratio rose progressively in culture. The isolation procedure and the duration of culture affected expression of specific P450s differently. CYP1A2, CYP2C9, and CYP2E1 mRNAs were not altered by cell isolation, and levels of CYP1A2 and CYP2C9 mRNA were also maintained for 8 days in culture, whereas CYP2E1 mRNA declined to 9% of values in fresh hepatocytes by day 8. CYP3A4 mRNA content was considerably decreased in freshly isolated hepatocytes compared with normal liver, and expression of this gene during the course of culture was more variable than that of the other P450s. Use of Williams' E medium considerably enhanced accumulation of CYP3A4 mRNA, compared with modified Waymouth 752/1 medium, but had a detrimental effect on levels of the other P450 mRNAs. Despite high levels of expression at the mRNA level, the microsomal protein contents of CYP1A2, CYP2C9, CYP2E1, and CYP3A4 declined progressively during the course of culture; this decline was most rapid for CYP3A4. These results confirm the potential of primary cultures of well-differentiated human hepatocytes for studies of P450 gene regulation in humans, but they also demonstrate that culture conditions are variables that must be carefully controlled when examining liver-specific gene expression in vitro. In particular, time in culture may variably affect expression of P450 enzyme changes at both the mRNA and protein levels.