The human equilibrative nucleoside transporter 3 (hENT3) was recently reported as a pH-dependent, intracellular (lysosomal) transporter capable of transporting anti-HIV dideoxynucleosides (ddNs). Because most anti-HIV ddNs (e.g. zidovudine, AZT) exhibit clinical mitochondrial toxicity, we investigated if hENT3 facilitates transport of anti-HIV ddNs into the mitochondria. Cellular fractionation and immunofluorescence microscopy studies in several human cell lines identified a substantial presence of hENT3 in the mitochondria with additional presence at the cell surface of two placental cell lines (JAR, JEG3). Mitochondrial or cell surface hENT3 expression was confirmed in human hepatocytes and placental tissues, respectively. Unlike endogenous hENT3, YFP-tagged hENT3 was partially directed to the lysosomes. Xenopus oocytes expressing N-terminal deleted hENT3 (expressed at the cell surface) showed pH-dependent interaction with several classes of nucleosides (anti-HIV ddNs, gemcitabine, fialuridine, ribavirin) that produce mitochondrial toxicity. Transport studies in hENT3 gene-silenced JAR cells showed significant reduction in mitochondrial transport of nucleosides and nucleoside drugs. Our data suggest that cellular localization of hENT3 is cell-type dependent and the native transporter is substantially expressed in mitochondria and/or cell surface. hENT3-mediated mitochondrial transport may play an important role in mediating clinically observed mitochondrial toxicity of nucleoside drugs. In addition, our findings that hENT3 is a mitochondrial transporter is consistent with the recent finding that mutations in the hENT3 gene causes an autosomal recessive disorder in humans called the H syndrome.