The human ATP1AL1-encoded protein (an alpha subunit of the human non-gastric H,K-ATPase) has previously been shown to assemble with the gastric H,K-ATPase beta subunit (gH,Kbeta) to form a functionally active ionic pump in HEK 293 cells. This pump has been found to be sensitive to both SCH 28080 and ouabain. However, the 86Rb+-influx mediated by the ATP1AL1-gH,Kbeta heterodimer in HEK 293 cells is at least 1 order of magnitude larger than the maximum ouabain-sensitive proton efflux detected in the same cells. In this study we find that the intracellular Na+ content in cells expressing ATP1AL1 and gH,Kbeta is two times lower than that in control HEK 293 cells in response to incubation for 3 h in the presence of 1 microM ouabain. Moreover, analysis of net Na+ efflux in HEK 293 expressing the ATP1AL1-gH,Kbeta heterodimer reveals the presence of Na+ extrusion activity that is not sensitive to 1 microM ouabain but can be inhibited by 1 mM of this drug. In contrast, ouabain-inhibitable Na+ efflux in control HEK 293 cells is similarly sensitive to either 1 microM or 1 mM ouabain. Finally, 86Rb+ influx through the ATP1AL1-gH,Kbeta complex is comparable to the 1 mM ouabain-sensitive Na+ efflux in the same cells. The data presented here suggest that the enzyme formed by ATP1AL1 and the gastric H,K-ATPase beta subunit in HEK 293 cells mediates primarily Na+,K+ rather than H+,K+ exchange.