Abstract In the presence of a specific ionic environment, sodium, potassium-adenosine triphosphatase (Na +,K +-ATPase) bound the inhibitor ouabain, inducing an apparent protein conformational change. Ouabain reversed a specific potassium-induced depression in fluorescence intensity and quantum yield of 1-anilino-8-naphthalene-sulfonic acid (ANS) and 2- p-toluidinyl-naphthalene-6-sulfonic acid (TNS) bound to a membrane preparation containing Na +,K +-ATPase isolated from guinea pig brain synaptic membranes (SM). The characteristics of the chromophore-bound SM were specifically changed when ouabain was added under the same conditions known to facilitate specific Na +,K +-ATPase-ouabain interaction. All changes followed a time course similar to that observed for ouabain binding. The life time and relaxation time of fluorescence of TNS bound-SM were also altered by ouabain under these ionic conditions suggesting an alteration in environment of the dye and/or Brownian movement of the probe-containing particles. These data are compatible with the suggestion that ouabain binding to Na +,K +-ATPase involves specific ligand-induced conformational changes. The inhibition of Na +,K +-ATPase by cardiac glycosides presumably results from these effects.