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蜑pyrimi-dine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Parasitic protozoa of the genus Leishmania are the etiological agents of leishmaniasis, a disease that affects an estimated 12 million people worldwide (1) and ranges from the disfiguring cutaneous form to fatal visceral leishmaniasis (2). Because current empirically identified drugs suffer from many deficiencies, including toxicity and resistance, it is important to identify unique biochemical targets that could be exploited for rational development of improved therapies. Perhaps the most striking metabolic discrepancy between parasites and their hosts is the purine pathway. Whereas most mammalian cells synthesize purines de novo, all parasitic protozoa studied to date are unable to synthesize purines (3) and consequently must rely on purine acquisition from their hosts for survival and growth. The first step in this salvage pathway involves the transport of these substrates across the parasite plasma membrane. Moreover, these purine transporters initiate the uptake of certain pyrazolopyrimidine analogs of hypoxanthine and inosine that are toxic to both Leishmania and Trypanosoma (4). These pyrazolopyrimidines, such as allopurinol, allopurinol riboside, and formycin B, are subsequently metabolized to the nucleotide level by the parasite metabolic machinery and incorporated into RNA, metabolic transformations that do not occur in mammalian cells (4). Both the essential nutritional function of these transporters and their roles in mediating the toxicities of well-characterized antiparasitic agents provide compelling rationale to study these membrane permeases at the molecular level.Biochemical and genetic studies have established that Leishmania donovani parasites express two distinct nucleoside transporters with nonoverlapping substrate specificities (5). One transporter mediates the uptake of adenosine and pyrimidine nucleosides and also transports tubercidin, a cytotoxic analog of adenosine, whereas the other transporter allows membrane permeation of guanosine, inosine, and formycin B (5). Parasites deficient in either or both transport activities have been isolated by mutagenesis with N-methyl-N袌-nitro-N-nitrosoguanidine followed by selection in tubercidin or formycin B (6). The availability of these null mutants provided a functional strategy for cloning genes encoding each of these nucleoside permeases.In the present study, we have transfected the adenosine蜑pyrimidine nucleoside transport-deficient TUBA5 cell line with a cosmid library containing inserts of L. donovani genomic DNA (7) and screened individual transfectants for restoration of tubercidin...