Type III Na+-coupled Pi cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue. They mediate electrogenic Pi cotransport when expressed in Xenopus oocytes. We studied the transport function of 3 isoforms: Xenopus PiT-1, human PiT-1 and PiT-2. Each clone gave robust Na+ dependent 32Pi uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization using two-electrode voltage clamp and 32P uptake. The dependency of the Pi-induced current on [Pi] was Michaelian and the dependency on [Na+] indicated weak cooperativity. The apparent Pi affinity constant showed a minimum in the pH range 6.2-6.8 of ~0.05 mM and increased to ~0.2 mM at pH 5.0 and pH 8.0. A 2:1 Na+:Pi stoichiometry was determined by dual 22Na, 32Pi uptake. Correlation of 32Pi uptake and net charge movement indicated that 1 charge was translocated per Pi. Changes in oocyte surface pH were consistent with transport of monovalent Pi. Based on the kinetics of substrate interdependency, we propose an ordered binding scheme of Na+:H2PO4-:Na+. Significantly, in contrast to type II Na+/Pi cotransporters, the transport inhibitor phosphonoformic acid (PFA) did not inhibit PiT-1 or PiT-2 activity.