A polyclonal antibody against the Na+,K(+)-ATPase holoenzyme was prepared. This antibody recognized native Na+,K(+)-ATPase and inhibited its activity. The peptide fragments corresponding to various regions of the Na+,K(+)-ATPase alpha-subunit, which were synthesized from the cDNA, were immunoprecipitated with the antibody, and the M32-D75, M158-D197 and M470-V552 fragments (the latter included K508, a putative ATP binding site) were identified as the epitopes. The M267-I442 fragment, which included a phosphorylation site at D376, and the C-terminal one-third of the alpha-subunit from M615 to the C-terminus, were not detected using this antibody. These results suggest that at least three regions of the Na+,K(+)-ATPase alpha-subunit, M32-D75, M158-D197 and M470-V552, cover its exposed regions, and that some of them are essential for ATPase activity.