The study of drug metabolism in cultured rat hepatocytes is hampered by the rapid loss of the expression of cytochrome P450 enzymes. Nevertheless, the activity of cytochrome P450 3A (CYP3A), one of the most important isoenzymes for drug metabolism, can be elevated by chemical inducers. In the present study, we investigated in cultured rat hepatocytes the induction of all four currently identified CYP3A isoforms by dexamethasone, and compared the results obtained in vitro with the induction profile of dexamethasone in vivo. To this end, CYP3A mRNA levels were quantified with a novel, radioactive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and CYP3A enzymatic activity was measured by a testosterone hydroxylation assay. In the RT-PCR assay, CYP3A isoforms were co-amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of radioactively labeled nucleotides. This resulted in an extremely sensitive and accurate determination of CYP3A expression levels, relative to those of GAPDH. Using this RT-PCR assay, it was found that the expression of all CYP3A isoforms in rat hepatocytes, cultured on a collagen matrix, was decreased by 80-90% within one day of cultivation. After addition of dexamethasone, at one day after isolation, CYP3A1 mRNA levels were elevated to levels comparable to those in freshly isolated hepatocytes within two days. In contrast, CYP3A2, CYP3A9, and CYP3A18 mRNA levels were not affected by dexamethasone treatment, and were hardly detectable after three days of cultivation. CYP3A enzymatic activity was also induced in cultured hepatocytes (approximately 6-fold) after addition of dexamethasone. In vivo, CYP3A1 mRNA levels increased 45-fold after dexamethasone administration. However, in contrast to the situation in cultured hepatocytes, CYP3A2 and CYP3A18 were also induced, albeit to a lesser extent (4- and 7-fold elevated mRNA levels, respectively). We conclude that the selective induction of CYP3A1 in dexamethasone-treated rat hepatocytes allows the study of biotransformation reactions by CYP3A1, without interference by any of the other CYP3A isoenzymes.