Two Na(+)-dependent nucleoside transporters implicated in adenosine and uridine transport in mammalian cells are distinguished functionally on the basis of substrate specificity: CNT1 is selective for pyrimidine nucleosides but also transports adenosine; CNT2 (also termed SPNT) is selective for purine nucleosides but also transports uridine. Both proteins belong to a gene family that includes the NupC proton/nucleoside symporter of E. coli. cDNAs encoding members of the CNT family have been isolated from rat tissues (jejunum, brain, liver; rCNT1 and rCNT2/SPNT) and, most recently, human kidney (hCNT1 and hSPNT1). Here, the molecular cloning and functional characterization of a CNT2/SPNT-type transporter from human small intestine are described. The encoded 658-residue protein (hCNT2 in the nomenclature) had the same predicted amino acid sequence as human kidney hSPNT1, except for a polymorphism at residue 75 (Arg substituted by Ser), and was 83 and 72% identical to rCNT2 and hCNT1, respectively. Sequence differences between hCNT2 and rCNT2 were greatest at the N-terminus. In Xenopus oocytes, recombinant hCNT2 exhibited the functional characteristics of a Na(+)-dependent nucleoside transporter with selectivity for adenosine, other purine nucleosides and uridine (adenosine and uridine K(m) app values 8 and 40 microM, respectively). hCNT2 transcripts were found in kidney and small intestine but, unlike rCNT2, were not detected in liver. Deoxyadenosine, which undergoes net renal secretion in humans, was less readily transported than adenosine. hCNT2 also mediated small, but significant, fluxes of the antiviral purine nucleoside analogue 2',3'-dideoxyinosine. hCNT2 is, therefore potentially involved in both the intestinal absorption and renal handling of purine nucleosides (including adenosine), uridine and purine nucleoside drugs. The gene encoding hCNT2 was mapped to chromosome 15q15.