Human intestinal Caco-2 cell monolayers grown in the presence of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) were used to test the hypothesis that drugs which interact with the apical efflux pump P-glycoprotein (Pgp) may enhance CYP3A4-mediated disappearance of substrates. 6beta-hydroxytestosterone production, a marker of CYP3A4 activity, was approximately 3- and 7-fold greater in 1,25(OH)2D3-treated cells compared to untreated cells when incubated with 50 and 500 microM testosterone, respectively, and was unaffected by the addition of digoxin to reduce Pgp activity. In the presence of digoxin, secretory transport of vinblastine and erythromycin, substrates for both Pgp and cytochrome P450 3A4 (CYP3A4), was significantly reduced, whereas absorptive transport was unaffected. In contrast, no directional transport of testosterone, a substrate for CYP3A4 only, was observed, either in the presence or absence of digoxin. Over 2 h, disappearance of erythromycin and vinblastine from the incubation medium was significantly greater from the basolateral than from the apical compartments. In the presence of digoxin, disappearance of both compounds from the basolateral, but not from the apical compartments, was significantly reduced. In contrast, disappearance of testosterone was unaffected by the addition of digoxin, demonstrating that the effect of digoxin on erythromycin and vinblastine disappearance was via inhibition of Pgp function, rather than on CYP3A4 activity. Thus, evidence is provided for Pgp/CYP3A4 co-substrates, Pgp potentiates CYP3A4-mediated drug disappearance during intestinal secretory detoxification.