The Na+/nucleoside cotransporter in rabbit choroid plexus differs from Na+/nucleoside cotransporters in other tissues in terms of substrate selectivity and stoichiometry. The overall goal of this study was to further characterize the kinetics of this system (N3). Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP and treated with valinomycin and K+. Na+/thymidine uptake at 30 s in the presence of an inside negative potential difference was significantly greater than in the absence of a potential difference. Na+/thymidine uptake was not significantly affected by replacing chloride with either thiocyanate or sulfate. The Km of Na+/guanosine uptake was 149, 85.2 and 30.5 microM in the presence of a 25, 50 and 100 mM Na+ gradient, respectively, whereas the Vmax was unaffected, suggesting that Na+ binds first to the cotransporter, then, the nucleoside. Therapeutically relevant base-modified nucleoside analogs, 5-fluorouridine, 2-chloroadenosine and 5-iododeoxyuridine, significantly inhibited Na+/thymidine uptake with IC50 values (mean +/- S.E.) of 12.0 +/- 2.3, 21.3 +/- 2.2 and 24.4 +/- 2.1 microM, respectively, whereas nucleoside analogs structurally modified on the ribose ring, 3'-azidothymidine, dideoxyinosine and dideoxycytidine (100 microM) did not. These studies suggest that Na+/nucleoside cotransport in the choroid plexus is electrogenic and is not dependent on chloride. This cotransporter, which is present in choroid plexus but not in renal brush-border membrane vesicles from rabbit, may play a role in the disposition of clinically relevant base-modified nucleoside analogs into and out of the brain.