The hypothesis was tested that the operation of an ATP-dependent export pump localized at the apical (brush border) surface of the intestinal epithelium may limit substrate absorption kinetics. Human intestinal Caco-2 cell-layers display saturable secretion of vinblastine from basal to apical surfaces (Km, 18.99 +/- 5.55 microM; Vmax, 1285.9 +/- 281.2 pmol cm-2 hr-1) that is inhibited by verapamil, consistent with the expression of the ATP-dependent P-glycoprotein drug efflux pump at the apical brush border membrane. Inhibition of P-glycoprotein by a variety of modulators (verapamil, 1,9-dideoxyforskolin, nifedipine, and taxotere) is associated with an increased vinblastine absorptive permeability. Vinblastine absorption displayed a nonlinear dependence upon luminal (apical) vinblastine concentration, and vinblastine absorption increased markedly at concentrations where vinblastine secretory flux was saturated (> 20 microM). Upon inhibition of P-glycoprotein by verapamil and 1,9-dideoxyforskolin, vinblastine absorption increased and was linearly dependent on vinblastine concentration. The limitation of P-glycoprotein substrate absorption by active ATP-dependent export via P-glycoprotein is discussed, together with the possibility that other classes of substrate may be substrates for different ATP-dependent export pumps.