Oligomycin inhibits Na(+),K(+)-ATPase activity by stabilizing the Na(+) occlusion but not the K(+) occlusion. To locate the binding domain of oligomycin on Na(+),K(+)-ATPase, the tryptic-digestion profile of Na(+),K(+)-ATPase was compared with the profile of Na(+) occlusion within the digested Na(+),K(+)-ATPase in the presence of oligomycin. The Na(+) occlusion profile is responsible for the digestion profile of the alpha-subunit, which is the catalytic subunit of the ATPase. The effect of oligomycin on chimeric Ca(2+)-ATPase activity was examined. The chimera used, in which the 163 N-terminal amino acids of chicken sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 were replaced with the 200 N-terminal amino acids of the chicken Na(+),K(+)-ATPase alpha1-subunit, partially retains the Na(+)-dependent characteristics of Na(+), K(+)-ATPase, because the chimeric Ca(2+)-ATPase activity is activated by Na(+) but inhibited by ouabain, a specific inhibitor of Na(+),K(+)-ATPase (Ishii, T., Lemas, M.V., Takeyasu, K., 1994, Proc. Natl. Acad. Sci. U. S. A., 91, 6103-6107). Oligomycin depressed the activation by Na(+) of the chimeric Ca(2+)-ATPase activity. These findings suggest that the 200 N-terminal amino acids of the Na(+), K(+)-ATPase alpha-subunit include a binding domain for oligomycin.