Treatment of a purified (NA+ + 5+)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na+ + k+)-atpase reaction inhibited more than the K+-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na+ + k+)-atpase reaction but not the K+-phosphatase reaction; however, treatment with digitonin made the K+-phosphatase reaction almost as sensitive to oligomycin as the (Na+ + k+)-atpase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K+-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K+-phosphatase reaction more than the (Na+ + k+)-atpase reaction). Both digitonin and Triton markedly increased the K0.5 for K+ as activator of the K+-phosphatase reaction, with little effect on the K0.5 for K+ as activator of the (Na+ + k+)-ATpase reaction. In contrast, increasing the K0.5 for K+ in the K+-phosphatase reaction by treatment of the enxyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K+-phosphatase reaction by ATP and increased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K0.5 for K+ in the K+-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na+ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na+ plus nucleotide.