Purpose. The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na+-dependent, purine-selective nucleoside transporter (SPNTint) and to study the interactions of nucleosides and nucleoside analogs with this transporter. Methods. Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNTint. Nucleoside transport activity was measured using [3H] inosine, [3H]uridine, [3H]-dideoxyinosine (ddl), and [3H]-2-chloro-2′-deoxyadenosine (2CdA) as model substrates. Results. Expression of SPNTint was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na+-stimulated uptake of [3H]inosine in cells transiently transfected with SPNTint was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na+-stimulated uptake of both inosine and uridine was saturable (Km = 28.1 ± 7.1 μM and 20.6 ± 5.6 μM, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddl (IC50 = 46 μM) and 2CdA (IC50 =.13 μM) also significantly inhibited the Na+-stimulated uptake of [3H]inosine. A Na+-stimulated uptake of [3H]2CdA was observed suggesting that 2CdA is also a permeant of SPNTint. Conclusions. HeLa cells transiently transfected with SPNTint represent a useful tool to study the kinetics and interactions of drugs with SPNTint.