Hepatic thiamine transport is thought to be a saturable, Na(+)- and energy-dependent process. However, the transport of this organic cation has not been examined in experimental models that allow direct characterization of carrier-mediated processes. Recently, a sinusoidal organic cation/H+ antiport was identified, using N1-methylnicotinamide as a marker. To determine whether thiamine is a substrate for this antiport, the characteristics of thiamine uptake were examined in rat liver basolateral membrane vesicles. An inwardly directed Na+ gradient had no effect on thiamine uptake as compared with an identical K+ gradient. An outwardly directed H+ gradient stimulated thiamine uptake as compared with pH-equilibrated conditions, and H(+)-dependent uptake was not the result of an H+ diffusion potential. Identical pH gradients stimulated uptake under voltage-clamped conditions, consistent with electroneutral thiamine/H+ exchange. Unlabeled intravesicular thiamine trans-stimulated [3H]thiamine uptake. Choline and imipramine cis-inhibited thiamine/H+ exchange; a series of other organic cations and thiamine analogues had no effect. Carrier-mediated [3H]thiamine uptake showed two saturable systems. In conclusion, a thiamine/H+ antiport is present on the sinusoidal membrane, distinct from Na+/H+ and NMN+/H+ exchange.