The Na(+)-K+ pump is thought to operate through a two-conformation (E1-E2) transport cycle in which the cation binding sites are accessible only from one side at a time. Using Na(+)-loaded Xenopus oocytes in which Na(+)-K+ pumps were overexpressed by injection of cRNA of the Xenopus Na(+)-K+ pump alpha-and beta-sub units, we observed a Na(+)-K+ pump-mediated (ouabain-sensitive) inward current in the absence of other transportable cations, except H+, in the external solution. This inward current was strongly inwardly rectifying, pH dependent, and larger at acid pH. Under conditions favoring a large ouabain-sensitive inward current, we observed a ouabain-sensitive intracellular acidification, and the amplitude of the acidification was significantly related to the ouabain-sensitive current, indicating that this current was carried by protons. The reversal potential of the ouabain-sensitive current was dependent on external pH as expected for a proton-conductive pathway. We conclude that in the absence of external K+ the Na(+)-K+ pump can mediate a large inward electrogenic transport of proton. This is most easily explained by the hypothesis that the E2 conformation of the Na(+)-K+ pump with cation binding sites exposed to the outside is accessible to protons from both sides and thus provides a channellike pathway for protons.