We previously demonstrated that the α−subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase β-subunit (βHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1−βHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1−βHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and βHK. Expression of either protein alone did not produce active ATPase. The effects of K⁺, Na⁺, pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1−βHK complex were analyzed. The Atp1al1−βHK complex was shown to exhibit significant ATPase activity in nominally K⁺-free medium. The addition of K⁺ stimulated the ATP hydrolysis up to 3-fold with K_m ∼116 μM K⁺. The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K_i values in K⁺-free medium of ∼64 μM and ∼93 μM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na⁺ with the apparent K_i of ∼24 mM in the absence of added K⁺ and with K_i within the range of 60−70 mM in the presence of ≥1 mM K⁺. Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1−βHK complex exhibits enzymatic properties of K⁺-dependent ATPase sensitive to ouabain, SCH 28080, and Na⁺. It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.