OBJECTIVE The purpose of this work was to determine whether P-glycoprotein (P-gp) modulates absorptive and secretory transport equally across polarized epithelium (i.e., Caco-2 cell monolayers) for structurally diverse P-gp substrates, a requirement for the use of the efflux ratio to quantify P-gp-mediated attenuation of absorption across intestinal epithelium. METHODS Studies were performed in Caco-2 cell monolayers. Apparent permeability (P(app)) in absorptive (P(app,AB)) and secretory (P(app,BA)) directions as well as efflux ratios (P(app,BA)/P(app,AB)) were determined for substrates as a function of concentration. Transport of these compounds (10 microM) was measured under normal conditions and in the presence of the P-gp inhibitor, GW918 (1 microM), to dissect the effect of P-gp on absorptive and secretory transport. Apparent biochemical constants of P-gp-mediated efflux activity were calculated for both transport directions. RESULTS Efflux ratios for rhodamine 123 and digoxin were comparable (approx. 10). However, transport studies in the presence of GW918 revealed that P-gp attenuated absorptive transport of digoxin by approx. 8-fold but had no effect on absorptive transport of rhodamine 123 (presumably because absorptive transport of rhodamine 123 occurs via paracellular route). The apparent Km for P-gp-mediated efflux of digoxin was > 6-fold larger in absorptive vs. secretory direction. For structurally diverse P-gp substrates (acebutolol, colchicine, digoxin, etoposide, methylprednisolone, prednisolone, quinidine, and talinolol) apparent Km was approximately 3 to 8-fold greater in absorptive vs. secretory transport direction, whereas apparent J(max) was somewhat similar in both transport directions. CONCLUSIONS P-gp-mediated efflux activity observed during absorptive and secretory transport was asymmetric for all substrates tested. For substrates that crossed polarized epithelium via transcellular pathway in both directions, this difference appears to be caused by greater apparent Km of P-gp-mediated efflux activity in absorptive vs. secretory direction. These results clearly suggest that use of efflux ratios could be misleading in predicting the extent to which P-gp attenuates the absorptive transport of substrates.