Choline can undergo both net secretion and net reabsorption by renal proximal tubules, but at physiological plasma levels net reabsorption occurs. During this process, choline enters the cells at the luminal side down an electrochemical gradient via a specific transporter with a high affinity for choline. It appeared likely that choline was then transported out of the cells against an electrochemical gradient at the basolateral membrane by countertransport for another organic cation. This possibility was examined by studying net transepithelial reabsorption and basolateral uptake and efflux of [14C]choline in isolated S2 segments of rabbit renal proximal tubules. Basolateral uptake, which was inhibited by other organic cations such as tetraethylammonium (TEA), appeared to occur by the standard organic cation transport pathway. However, the addition of TEA to the bathing medium not only failed to trans-stimulate net transepithelial reabsorption and basolateral efflux of [14C]choline but it actually inhibited transepithelial reabsorption by @60%. The results do not support the presence of a countertransport step for choline against an electrochemical gradient at the basolateral membrane. Instead, they suggest that choline crosses this membrane by some form of carrier-mediated diffusion even during the reabsorptive process.