Recently, we demonstrated the presence of a Na(+)-nucleoside cotransport mechanism that transports both purine and pyrimidine nucleosides in human renal brush-border membrane vesicles (BBMV) (Gutierrez et al. (1992) Biochim. Biophys. Acta 1105, 1-9). The objective of this study was to further elucidate the characteristics of this cotransport system in terms of electrical potential sensitivity, stoichiometry and substrate selectivity with respect to nucleoside analogs. In BBMV from human kidney, Na(+)-thymidine uptake was stimulated by an inside negative potential difference created by K+ and valinomycin. A hyperbolic relationship between initial rate of uridine uptake and Na+ concentration was obtained suggesting a Na(+)-nucleoside coupling stoichiometry of 1:1. Our previous study had demonstrated that the pyrimidines, thymidine, cytidine, and uridine and the purines, adenosine, 2'-deoxyadenosine, and guanosine, but not inosine and formycin B, were substrates of this system. To further define the substrate selectivity of the transporter, the interaction of the drugs, 2-chloroadenosine (2-ClAdo), 5-fluorouridine (5-FUrd) and 5-iodo-2'-deoxyuridine (5-IdUrd), nucleoside analogs that are modified on the base moiety was studied. The three compounds inhibited Na(+)-thymidine uptake in the vesicles via a competitive mechanism. The IC50 values for 2-ClAdo, 5-FUrd and 5-IdUrd were 75, 49, and 16 microM, respectively. In addition, 5-IdUrd trans-stimulated the initial uptake of thymidine into the vesicles suggesting that the two compounds share the same transporter. Collectively, these data suggest that Na(+)-nucleoside transport in the human renal brush-border membrane is an electrogenic process and that the kidney may play a role in the disposition and targeting of clinically important nucleoside analogs.