The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1ยฑ2 mM) in plastic ยฏasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 mM 1a,25-dihydroxyvitamin D 3 to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213 AE 54.35%) compared to that of parent cells (100 AE 6.05%). Furthermore, they retained signiยฎcantly less [ 3 H]vincristine sulphate (p < 0.05), a P-gp substrate, after efยฏux (272.89 AE 11.86 fmol/mg protein) than the parent cells (381.39 AE 61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1a,25-dihydroxyvitamin D 3 treatment was quantiยฎed to be 76.2 AE 7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0 AE 20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0 AE 20.0 to 1057.0 AE 46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics. ร 2002