Expression screening in Xenopus oocytes was used to isolate a cDNA from rat jejunal epithelium encoding a Na(+)-dependent nucleoside transport protein (named cNT1). The cDNA sequence of cNT1 predicts a protein of 648 amino acids (relative molecular mass 71,000) with 14 potential transmembrane domains. Data base searches indicate significant sequence similarity to the NUPC proton/nucleoside symporter of Escherichia coli. There is no sequence similarity between cNT1 and proteins of mammalian origin. Functionally, cNT1 exhibited the transport characteristics of the nucleoside transport system cit (selective for pyrimidine nucleosides and adenosine) and accepted both 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) as permeants (Km = 0.49 and 0.51 mM, respectively). The demonstration of transport of AZT by cNT1 expressed in Xenopus oocytes provides the first direct evidence that AZT enters cells by transporter-mediated processes, as well as by passive diffusion. Consistent with the tissue distribution of system cit transport activity, transcripts for cNT1 were detected in kidney as well as jejunum. cNT1 therefore belongs to a potential new gene family and may be involved in the intestinal absorption and renal handling of pyrimidine nucleoside analogs used to treat acquired immunodeficiency syndrome (AIDS).