The Na⁺-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. Here, we have used the two-electrode voltage-clamp technique to investigate rat CNT2 transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates K_0.5 was in the low-range µM (12-34) and voltage-independent for hyperpolarizing membrane potentials, whereas I_max was voltage-dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. The lack of the 2'-hydroxyl group decreased the affinity but increased the I_max. Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I_max. Fludarabine and formycin B were also transported with higher I_max than uridine and moderate affinity (102±10 and 66±6 µM respectively). Analysis of the presteady-state currents revealed a V_0.5 of ~ - 39 mV and a z of ~ -0.8. K_0.5 for Na⁺ was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge-associated showed a ratio of two positive inward charges per nucleoside suggesting a stoichiometry of two Na⁺/nucleoside. This discrepancy in the number of Na⁺ molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na⁺ binding sites.