This study was undertaken to examine the effect of ethanol on Na⁢-dependent phosphate (Na⁢-Pi) uptake in opossum kidney (OK) cells, an established renal proximal tubular cell line. Ethanol inhibited Na⁢-dependent component of phosphate uptake in a dose-dependent manner with I50 of 8.4%, but it did not affect Na⁢-independent component. Similarly, ethanol inhibited Na⁢-dependent uptakes of glucose and amino acids (AIB, glycine, alanine, and leucine). Microsomal Na⁢-K⁢-ATPase activity was not significantly altered when cells were treated with 8% ethanol. Kinetic analysis showed that ethanol increased Km without a change in Vmax of Na⁢-Pi uptake. Inhibitory effect of n-alcohols on Na⁢-Pi uptake was dependent on the length of the hydrocarbon chain, and it resulted from the binding of one molecule of alcohol, as indicated by the Hill coefficient (n) of 0.8-1.04. Catalase significantly prevented the inhibition, but superoxide dismutase and hydroxyl radical scavengers did not alter the ethanol effect. A potent antioxidant DPPD and iron chelators did not prevent the inhibition. Pyrazole, an inhibitor of alcohol dehydrogenase, did not attenuate ethanol-induced inhibition of Na⁢-Pi uptake, but it prevented ethanol-induced cell death. These results suggest that ethanol may inhibit Na⁢-Pi uptake through a direct action on the carrier protein, although the transport system is affected by alterations in the lipid environment of the membrane.