Plasma membrane vesicles isolated from human platelets accumulate 5-hydroxytryptamine when an electrical potential (interior negative) or an Na+ gradient (out greater than in) is imposed across the vesicle membrane. Kinetic studies reveal a Km of 0.5 micronM for the transport process. Uptake is inhibited strongly by tricyclic antidepressants and by ionophores such as gramicidin which catalyze transmembrane exchange of Na+ for K+. Transport is absolutely dependent upon external Na+ and Cl- and is only mildly, if at all, inhibited by reserpine, cinanserin, ouabain, or arsenate. Experiments are presented which suggest that a single positive charge crosses the vesicle membrane with each molecule of 5-hydroxytryptamine. The results provide direct evidence for Na+-coupled active 5-hydroxytryptamine transport by the platelet plasma membrane.