Members of the SLC20 family or type III Na(+) -coupled P(i) cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P(i) homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P(i) cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na(+)-dependent (32)P(i) uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li(+) substituted for Na(+). The dependence of the P(i)-induced current on P(i) concentration was Michaelian, and the dependence on Na(+) concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P(i) affinity constant showed a minimum in the pH range 6.2-6.8 of approximately 0.05 mM and increased to approximately 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual (22)Na-(32)P(i) uptake and suggested a 2:1 Na(+):P(i) stoichiometry. A correlation of (32)P(i) uptake and net charge movement indicated one charge translocation per P(i). Changes in oocyte surface pH were consistent with transport of monovalent P(i). On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na(+):H(2)PO(4)(-):Na(+). Significantly, in contrast to type II Na(+)-P(i) cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity.