The aim of the present study was to characterize membrane transport mechanisms of mizoribine in the intestinal epithelial cells. We evaluated the contribution of Na⁺-dependent and -independent membrane transporters to mizoribine absorption in the rat intestine using an in situ closed loop method. In addition, we evaluated the effects of structurally related compounds, extracellular Na⁺ concentrations, and an inhibitor of Na⁺-independent equilibrative nucleoside transporter, nitrobenzylmercaptopurine ribonucleoside (NBMPR), on the uptake of mizoribine in human intestinal epithelial LS180 cells. In the presence and also absence of Na⁺ in rat intestinal loops, more than 60% of the administered dose (50 μg at the concentration of 100 μg/ml=386 μM) of mizoribine was absorbed in 40 min. In the LS180 cells, ribavirin and inosine reduced the uptake of 400 μM mizoribine with the increasing concentrations (from 5 to 50 mM) of the inhibitors. The cellular uptake of mizoribine in the absence of extracellular Na⁺ decreased to 72.7% of the uptake in the presence of extracellular Na⁺, whereas 100 μM NBMPR decreased the uptake of mizoribine markedly to 34.7% of that without NBMPR. These findings suggest that Na⁺-independent nucleoside transporters are largely responsible for absorption of mizoribine in the intestine.