We have characterized the kinetics of substrate transport in the renal type II human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and presteady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of substrate (³²P_i, ²²Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1, and that the preferred transported Pi species is HPO₄^2-. We then probed the complex interrelationship of substrate, pH and voltage in the NaPi-IIa transport cycle by analysing both steady-state and presteadystate currents. Steady-state current measurements show that the apparent HPO₄^2- affinity is voltage-dependent, and that this voltage dependency is abrogated by lowering the pH or the Na⁺ concentration. In contrast, the voltage dependency of the apparent Na⁺ affinity increased when pH was lowered. Presteady-state current analysis shows that Na⁺ ions bind first and influence the preferred orientation of the transporter in the absence of P_i. Presteady-state charge movement was partially suppressed by complete removal of Na⁺ from the bath, by reducing extracellular pH (both in the presence and absence of Na⁺), or by adding P_i (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HPO₄^2- binding and proton modulation of the first Na⁺ binding step.