The interaction of ATP with the phosphoenzyme of Na(+),K(+)-ATPase from pig kidney, rabbit kidney, and shark rectal gland was investigated using the voltage-sensitive fluorescent probe RH421. In each case, ATP concentrations >or=100 microM caused a drop in fluorescence intensity, which, because RH421 is sensitive to the formation of enzyme in the E2P state, can be attributed to ATP binding to the E2P phosphoenzyme. Simulations of the experimental behavior using kinetic models based on either a monomeric or a dimeric enzyme mechanism yielded a K(d) for ATP binding in the range 140-500 muM. Steady-state activity measurements and independent measurements of the phosphoenzyme level via a radioactive assay indicated that ATP binding to E2P causes a deceleration in its dephosphorylation when acting in the Na(+)-ATPase mode, i.e., in the absence of K(+) ions. Both the ATP-induced drop in RH421 fluorescence and the effect on the dephosphorylation reaction could be attributed to an inhibition of dissociation from the E2P(Na(+))(3) state of the one Na(+) ion necessary to allow dephosphorylation. Stopped-flow studies on the shark enzyme indicated that the ATP-induced inhibition of dephosphorylation is abolished in the presence of 1 mM KCl. A possible physiological role of allosteric binding of ATP to the phosphoenzyme could be to stabilize the E2P state and stop the enzyme running backward, which would cause dissipation of the Na(+) electrochemical potential gradient and the resynthesis of ATP from ADP. ATP binding to E2P could also fix ATP within the enzyme ready to phosphorylate it in the subsequent turnover.