To investigate whether nongastric H⁺-K⁺-ATPases transport Na⁺ in exchange for K⁺ and whether different β-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H⁺-K⁺-ATPase, ATP1al1 (AL1), and of the Na⁺-K⁺-ATPase α₁-subunit (α₁) expressed in Xenopus oocytes, with different β-subunits. Our results show that βHK and β₁-NK can produce functional AL1/β complexes at the oocyte cell surface that, in contrast to α₁/β₁ NK and α₁/βHK complexes, exhibit a similar apparent K⁺ affinity. Similar to Na⁺-K⁺-ATPase, AL1/β complexes are able to decrease intracellular Na⁺ concentrations in Na⁺-loaded oocytes, and their K⁺ transport depends on intra- and extracellular Na⁺ concentrations. Finally, controlled trypsinolysis reveals that β-isoforms influence the protease sensitivity of AL1 and α₁ and that AL1/β complexes, similar to the Na⁺-K⁺-ATPase, can undergo distinct K⁺-Na⁺- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H⁺-K⁺-ATPase interacts with and transports Na⁺ in exchange for K⁺ and that β-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na⁺-K⁺-ATPase α-subunit.