The effects of depolarizing concentrations of potassium chloride on the modulation of [3H]hemicholinium-3 binding sites and high affinity choline uptake were examined in vitro. When rat striatal slices were incubated in Krebs buffer for 20 min, [3H]hemicholinium-3 binding sites diminished to 60% of binding measured in fresh un-incubated tissue, and remained stable for 60 min. Upon addition of Krebs buffer containing 40 mM KCl, the number of binding sites increased during a 20 min period, and remained stable for 40 min. Changes in [3H]hemicholinium-3 binding sites closely paralleled changes in high affinity choline uptake. Scatchard analysis revealed that changes in binding result from alterations in the number of binding sites (Bmax), and not in the affinity (KD). These results suggest that neuronal depolarization rapidly alters the velocity of choline transport into cholinergic neurons by increasing the number of available carriers.