All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine/pyrimidine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Two such genes, LdNT1.1 and LdNT1.2, have been sequenced and shown to encode deduced polypeptides with significant sequence identity to the human facilitative nucleoside transporter hENT1. Hydrophobicity analysis of the LdNT1.1 and LdNT1.2 proteins predicted 11 transmembrane domains. Transfection of the adenosine/pyrimidine nucleoside transport-deficient TUBA5 parasites with vectors containing the LdNT1.1 and LdNT1.2 genes confers sensitivity to the cytotoxic adenosine analog tubercidin and concurrently restores the ability of this mutant line to take up [3H]adenosine and [3H]uridine. Moreover, expression of the LdNT1.2 ORF in Xenopus oocytes significantly increases their ability to take up [3H]adenosine, confirming that this single protein is sufficient to mediate nucleoside transport. These results establish genetically and biochemically that both LdNT1 genes encode functional adenosine/pyrimidine nucleoside transporters.