The aim of this study was to find a cell culture model of the intestinal epithelium for use in studies of CYP3A4-mediated first-pass metabolism of drugs and also for studies of the interplay between CYP3A4 metabolism and P-glycoprotein efflux. For this purpose, the expression of CYP3A4, CYP3A5, and MDR1 mRNA was studied in three cell lines of the normal human intestinal epithelium and three transformed cell lines of colonic (Caco-2) origin. Surprisingly, only transformed cell lines were induced by 1alpha,25-dihydroxy vitamin D3 (D3) to express high amounts of CYP3A4. In contrast to the original findings for this model, the monolayer integrity was maintained during D3 treatment. Levels of CYP3A mRNA expression in Caco-2 and TC7 cells differed dramatically. The highest levels of CYP3A4 and lowest levels of CYP3A5 mRNA expression were observed in D3 treated Caco-2 cells of high passage numbers, resulting in a CYP3A4/3A5 expression ratio greater than fourfold higher than that seen in TC7 cells. Functional studies, using the CYP3A probe testosterone, showed that CYP3A activity was completely absent only in uninduced Caco-2 cells. After D3 induction, high levels of the metabolite were produced in both cell lines (149.4 +/- 12.3 and 86.5 +/- 3.8 pmol 6beta-OH testosterone/min/mg cellular protein from 75 microM testosterone in Caco-2 and TC7 cells, respectively). The maximum velocity (Vmax) and the apparent Michaelis constant (Km) for the 6beta-hydroxylation of testosterone by CYP3A4 in intact Caco-2 monolayers were similar to those obtained from human intestinal microsomes. Only minor changes in P-glycoprotein activity were observed when the metabolically stable P-glycoprotein substrate celiprolol was used. In conclusion, these results show that the features of the generally available Caco-2 cell line from American Type Culture Collection make it suitable for studies of CYP3A4-mediated first-pass metabolism and also for studies of the interplay between CYP3A4 and drug efflux mechanisms.