H2O2 dose dependently inhibited dopamine uptake in PC12 cells and in striatal synaptosomes. Treatment with H2O2 resulted in a reversible reduction in Vmax, with no effect on its Km value. This suppressive effect of H2O2 could be relieved by reducing agents (dithiothreitol and cysteine). Furthermore, an oxidizer (dithiodipyridine) also markedly suppressed the dopamine transporter (DAT). Oxidative stress therefore might contribute to the action of H2O2. H2O2 appeared to modify DAT at both extracellular and intracellular sites because cumene-H2O2 (a radical generator mostly restricted to plasma membranes) at high concentrations also slightly suppressed DAT activity and the intracellular overexpression of catalase ameliorated the inhibitory effect of H2O2. Internalization was unlikely to be involved because concanavalin A, which blocked endocytosis, did not prevent the H2O2-evoked inhibition of DAT activity. Interestingly, H2O2 treatment evoked a Ca2+ influx in PC12 cells. Moreover, removal of external calcium by EGTA or reduction in the intracellular calcium level using BAPTA-AM reversed the inhibitory effect of H2O2. Conversely, depletion of intracellular calcium stores using thapsigargin did not affect the reduction in DAT activity by H2O2. Collectively, our results indicate that the DAT, one of the most important proteins controlling the dopaminergic system, is also a redox sensor. In addition, H2O2 might suppress the DAT by a Ca2+-dependent oxidative pathway.