The human renal Na-PO4 cotransporter gene NaPi-3 was expressed in human embryonic kidney HEK-293 cells, and the transport characteristics were measured in cells transfected with a vector containing NaPi-3 or with the vector alone (sham transfected). The initial rate of 32PO4 influx had saturation kinetics for external Na and PO4 with K1/2Na of 128 mM (PO4 = 0.1 mM) and K1/2PO4 of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfected cells expressing the transporter. Transfection had no effect on the Na-independent 32PO4 influx, but transfection increased Na-dependent 32PO4 influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenate inhibited flux with an inhibition constant of 0.4 mM. The phosphate transport in sham- and NaPi-3-transfected cells has nearly the same temperature dependence in the absence and presence of extracellular Na. The Na-dependent phosphate flux decreased with pH in sham-transfected cells but was pH independent in transfected cells. The Na-dependent 32PO4 influx was inhibited by p-chloromercuriphenylsulfonate, phosphonoformate, phloretin, vanadate, and 5-(N-methyl-N-isobutyl)-amiloride but not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior of NaPi-3 homologues in the renal tubule of other species and, thus, demonstrate the fidelity of this transfection system for the study of this protein. Commensurate with the increased functional expression, there was an increase in the amount of NaPi-3 protein by Western analysis.