Type IIa/b Na(+)-coupled inorganic phosphate cotransporters (NaPi-IIa/b) are considered to be exclusively Na(+) dependent. Here we show that Li(+) can substitute for Na(+) as a driving cation. We expressed NaPi-IIa/b in X. laevis oocytes and performed two-electrode voltage-clamp electrophysiology and uptake assays to investigate the effect of external Li(+) on their kinetics. Replacement of 50% external Na(+) with Li(+) reduced the maximum transport rate and the rate-limiting plateau of the Pi-induced current began at less hyperpolarizing potentials. Simultaneous electrophysiology and 22Na uptake on single oocytes revealed that Li(+) ions can substitute for at least one of the 3 Na(+) ions necessary for cotransport. Presteady-state assays indicated that Li(+) ions alone interact with the empty carrier, however the total charge displaced was 70% of that with Na(+) alone, or when 50% of the Na(+) was replaced by Li(+). If Na(+) and Li(+) were both present, the midpoint potential of the steady-state charge distribution was shifted towards depolarizing potentials. The charge movement in the presence of Li(+) alone reflected the interaction of one Li(+) ion, in contrast to 2 Na(+) ions when only Na was present. We propose an ordered binding scheme for cotransport in which Li(+) competes with Na+ to occupy the putative 1st cation interaction site, followed by the cooperative binding of one Na(+) ion, one divalent Pi anion and a 3rd Na(+) ion to complete the carrier loading. With Li(+) bound, the kinetics of subsequent partial reactions were significantly altered. Kinetic simulations of this scheme support our experimental data.