The objective of the present study was to elucidate the mechanisms of intestinal transport of bis(12)-hupyridone (B12H) to predict its oral bioavailability. The effect of the B12H concentration and the contribution of the drug efflux transporters, P-glycoprotein (P-gp or ABCB1) and multidrug resistance-associated proteins (MRPs or ABCC) on B12H absorption were measured and evaluated using the human intestinal epithelial Caco-2 cell monolayer in the presence of transporter inhibitors. The results indicated that B12H was absorbed in a dose-dependent manner at concentrations ranging from 132 to 264 mu M. However, only apical efflux was observed in the directional transport studies for B12H below 88 mM (P(app)(AP-to-BL): virtually zero; P(app)(BL-to-AP): 1.591 +/- 0.071 x 10(-5) cms(-1)). P-gp and mixed P-gp/MRP inhibitors significantly increased the absorptive transport (P(app)(AP-to-BL)) to 0.619 +/- 0.018 x 10(-5) and 0.608 +/- 0.025 x 10(-5) cms(-1), respectively, while decreasing secretory transport (P(app)(BL-to-AP)) by > 75%. A multiple-MRP inhibitor, probenecid, increased the P(app)(AP-to-BL) to 0.329 +/- 0.015 x 10(-5) cms(-1) while decreasing the P(app)(BL-to-AP) by 50%. Another multiple-MRP inhibitor, indomethacin, only modestly decreased the P(app)(BL-to-AP) by similar to 30% and had no effect on the absorptive transport (P(app)(AP-to-BL): virtually zero). In addition, the effect of various pharmaceutical excipients (e.g. Pluronic F-68, Tween-80 and Brij-35) on B12H transport was determined and compared. Among them, Brij-35 effectively enhanced B12H absorption at a concentration lower than its critical micelle concentration (CMC, 60 mu M). Therefore, Brij-35 can be used as a potential enhancer to improve intestinal absorption of B12H for oral administration.