Glycerophosphorylcholine (GPC) plays an important role in the osmoregulation of the renal inner medulla. Under hyperosmotic conditions, a striking increase in cellular GPC content is observed. In order to characterize the cellular events involved in GPC metabolism, we have studied the uptake of choline, a precursor of GPC, by freshly isolated rat inner medullary collecting duct (IMCD) cells at 300 mosmol/l. Choline uptake occurred by a single transport system with an apparent affinity (Km) of 80 microM and a maximal velocity (Vmax) of 120 pmol/microliter cell water/min. Hemicholinium-3, ethanolamine and N,N-dimethylethanolamine were potent inhibitors, but betaine had no effect. Choline uptake was not altered by the replacement of Na+ with N-methylglucamine+, suggesting a sodium-independent process. Addition of 50 mM KCl to the incubation medium to reduce the cell membrane potential inhibited choline uptake by 19 +/- 4% after 10 min. Increasing the extracellular osmolarity to 600 or 900 mosmol/l had no effect on the kinetic parameters of choline uptake. These results suggest that choline uptake into IMCD cells occurs by a sodium-independent transport system driven by the inside negative cell membrane potential. Furthermore, the increase in the GPC content under hyperosmotic conditions is not associated with increased activity of the transport systems of biosynthetic precursors.