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The mechanism whereby oligomycin occludes Na+ within Na/K-ATPase was investigated to study Na+ and K+ transport mechanisms. Oligomycin stimulated Na+ binding to Na/K-ATPase but inhibited Na-K and Na-Na exchange. The oligomycin concentration required to stimulate Na+ binding to half-maximal was 4.5 microM, which was close to the concentration that reduced Na-Na and Na-K exchange and ATPase activity to half-maximal, suggesting that Na/K-ATPase possesses an oligomycin binding site responsible for stimulating Na+ binding and reducing ion exchange and ATPase activity. In contrast, neither K+ binding nor K+ transport was affected by oligomycin. Limited tryptic digestion of Na/K-ATPase showed that, unlike Na+, K+, and ouabain, oligomycin treatment did not result in a specific digestion pattern. Oligomycin appeared to inhibit ouabain binding in a noncompetitive manner, whereas it did not affect ATP binding. Na/K-ATPase isoforms with low and high sensitivities to ouabain were equally sensitive to oligomycin. These results suggest that the oligomycin binding site is located on the extracellular side of Na/K-ATPase, at a different position from the ouabain binding site, and this antibiotic did not induce a conformational change of Na/K-ATPase. We propose that oligomycin interacts with the Na+ occlusion site from the extracellular side of Na/K-ATPase, which delays Na+ release to the extracellular side without inducing a conformational change, suggesting that the pathways responsible for Na+ and K+ transport differ.