A pH difference (acid inside) across the platelet plasma membrane increases both the rate and extent of serotonin accumulation inside plasma membrane vesicles. Even in the absence of other transmembrane ion gradients, this pH difference (delta pH) serves as the sole driving force for serotonin accumulation, leading to a serotonin concentration 18-fold higher inside the vesicle. This process requires Na+ and is blocked by imipramine, indicating that it is mediated by the serotonin transporter. At physiological pH, internal K+ is counter-transported with serotonin, and high internal K+ stimulates transport maximally. Internal K+ also blocks the delta pH stimulation of serotonin transport. Conversely, low internal pH (5.6) inhibits the ability of internal K+ to stimulate transport. This apparent competition between K+ and protons suggests that delta pH drives serotonin accumulation through counter-transport with protons, and that serotonin is transported in its cationic form.