We previously reported that the human Na(+)/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5'-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine in hCNT1 function has not been addressed either. In the present work we have used the two-electrode voltage clamp technique to investigate hCNT1 transport mechanism and study the kinetic properties of adenosine as an inhibitor of hCNT1. We show that hCNT1 exhibits presteady-state currents that disappear upon the addition of adenosine or uridine. Adenosine, a purine nucleoside described as a substrate of the pyrimidine-preferring transporters, is not a substrate of hCNT1 but a high affinity blocker able to inhibit uridine-induced inward currents, the Na(+)-leak currents, and the presteady-state currents, with a K(i) of 6.5 microM. The kinetic parameters for uridine, gemcitabine, and 5'-deoxy-5-fluorouridine were studied as a function of membrane potential; at -50 mV, K(0.5) was 37, 18, and 245 microM, respectively, and remained voltage-independent. I(max) for gemcitabine was voltage-independent and accounts for approximately 40% that for uridine at -50 mV. Maximal current for 5'-DFUR was voltage-dependent and was approximately 150% that for uridine at all membrane potentials. K(0.5)(Na(+)) for Na(+) was voltage-independent at hyperpolarized membrane potentials (1.2 mM at -50 mV), whereas I(max)(Na(+)) was voltage-dependent, increasing 2-fold from -50 to -150 mV. Direct measurements of (3)H-nucleoside or (22)Na fluxes with the charge-associated revealed a ratio of two positive inward charges per nucleoside and one Na(+) per positive inward charge, suggesting a stoichiometry of two Na(+)/nucleoside.