The overall goal of this study was to determine the mechanisms by which nucleobases are transported in the choroid plexus. Choroid plexus tissue slices were obtained from the lateral ventricles of rabbit brains and depleted of ATP with 2,4-dinitrophenol. In the presence of an initial inwardly directed Na+ gradient, hypoxanthine accumulated in the tissue slices against a concentration gradient. Na(+)-stimulated hypoxanthine uptake was saturable with a Km of 31.1 +/- 9.71 microM and a Vmax of 2.69 +/- 0.941 nmol/g/s (mean +/- S.E.). Na(+)-stimulated hypoxanthine uptake was inhibited by (100) microM naturally occurring purine and pyrimidine nucleobases (adenine, cytosine, guanine, hypoxanthine, thymine, uracil, and xanthine) as well as by the nucleoside analog, dideoxyadenosine. The stoichiometric coupling ratio between Na+ and hypoxanthine was 1.7:1. The data demonstrate the presence of a novel Na(+)-dependent nucleobase transporter in the choroid plexus, which is distinct from the previously described Na(+)-nucleoside transporter in choroid plexus and from Na(+)-dependent nucleobase transporters in other tissues in terms of its kinetics, substrate selectivity, and Na(+)-nucleobase stoichiometry. This transporter may play a role in the targeting of both salvageable nucleobases and therapeutic nucleoside analogs to the central nervous system.