Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase alpha(2)- and Na,K-ATPase beta(2)-subunits; Bufo Na,K-ATPase alpha(1)- and Na,K-ATPase beta(2)-subunits; and Bufo Na,K-ATPase beta(2)-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 microM ouabain. Functional expression was confirmed by measuring (86)Rb uptake. PTX (5 nM: ) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo beta(2)-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase alpha(1)-subunit and Na,K-ATPase beta(1)-subunit; rat Na,K-ATPase alpha(2)-subunit and Na,K-ATPase beta(2)-subunit; and rat Na,K-ATPase beta(1)- or Na,K-ATPase beta(2)-subunit alone. Measurement of increases in (86)Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKalpha(1)/NKbeta(1) and NKalpha(2)/NKbeta(2). Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKalpha(1)/NKbeta(1) exposed to 100 nM PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKbeta(1)- or rat NKbeta(2)-subunit alone. However, in HeLa cells expressing rat NKalpha(2)/NKbeta(2), outward current was observed after pump activation by 20 mM K(+) and a large membrane conductance increase occurred after 100 nM PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.