This study was performed to characterize the mechanism of choline transport into human keratinocytes. Uptake of [3H]choline was measured both in the HaCaT cell line and in native keratinocytes. Uptake in HaCaT cells was linear with time at least up to 10 min. There was little dependence of choline transport on sodium. Choline uptake was slightly stimulated by extracellular H+ with the pH optimum being 7.5. The uptake rate was saturable and indicated participation of a single transport system (Kt = 14.8 +/- 1.0 micro M, Vmax = 1.0 +/- 0.01 nmol per 10 min per mg protein). The choline uptake into HaCaT cells was inhibited by unlabeled choline, hemicholinium-3, and acetylcholine. The prototypical organic cation tetraethylammonium showed very little affinity for the choline uptake system in these cells. Several cationic drugs such as diphenhydramine, clonidine, and atropine also interacted with the transport system. Choline uptake in normal keratinocytes was very similar to that in HaCaT cells with respect to substrate specificity and affinity. We conclude that keratinocytes express a Na+ independent, high-affinity choline transport system. This system accepts many pharmacologically important organic cations as substrates. It is similar or identical to the choline carrier described in intestinal epithelial cells and in endothelial cells of the blood-brain barrier. The choline carrier seems to have relevance not only for the uptake of cationic drugs into the keratinocytes but also for the biosynthesis of skin lipids.