Choline, an endogenous quaternary ammonium ion, is transported into the liver by both saturable and nonsaturable processes. The objective of the present investigation was to determine the driving force(s) for uptake of choline in rat liver basolateral membrane (blLPM) and canalicular membrane (cLPM) vesicles. Choline is transported into an osmotically sensitive intravesicular space in both blLPM and cLPM. Uptake of [3H]choline into both blLPM and cLPM exhibited temperature dependence (0 degree C vs. 37 degrees C). A valinomycin-induced inside-negative K+ diffusion potential significantly stimulated initial uptake of [3H]choline in both vesicles. Choline uptake in blLPM and cLPM was not stimulated in the presence of an inwardly directed sodium gradient or an outwardly directed H+ gradient, and ATP did not enhance choline uptake in cLPM. Choline itself and structurally similar derivatives, such as hemicholinium-3 and succinylcholine, inhibited [3H]choline uptake 11 to 92% (at 10-fold higher concentrations) in blLPM and cLPM. Other cations, including N1-methylnicotinamide, thiamine and d-tubocurarine, and cardioglycosides did not inhibit choline transport in either vesicle preparation. In addition, [3H]choline uptake into both blLPM and cLPM was enhanced when vesicles were preloaded with nonradiolabeled choline (trans-stimulation). Kinetic studies indicated that choline was transported into blLPM by both saturable and passive processes and into cLPM predominantly by a saturable process. These results suggest that the transport of choline is likely mediated by a potential-sensitive conductive pathway in both blLPM and cLPM. The electrogenic pathway in cLPM may play a role in the reabsorption of choline from bile.