The effects of ethanol, n-butanol, n-hexanol and n-octanol on lipid-protein interactions in sarcoplasmic reticulum vesicles (SRV) are investigated using the C-14 nitroxide spin-labeled phosphatidylcholine. n-Alkanols, which activate the Ca(2+)-dependent ATPase of sarcoplasmic reticulum but decrease net Ca2+ uptake by the vesicles, are shown to affect the lipids interacting with the protein surface. Spectral analysis revealed that increasing concentrations of the alcohols progressively displace and mobilize lipids from the lipid/protein interface. For butanol, hexanol and octanol maximally activated SRV, 23 to 30% of the protein-interacting lipids are displaced. Thus, the displacement of more than 30% of the annular lipids by these alkanols cause inhibition of the enzyme. The motional properties of the labels that remain restricted by the protein surface are unaffected by the alcohols. The degree of mobilization attained by the labels displaced from the interface is much greater than that observed in alcohol-treated dispersions of extracted lipids. We propose that the alcohol molecules interfere with the protein-lipid interactions creating fluid clusters around the proteins. These fluidized regions would affect the enzyme conformation, perturbing its function. Fluidized annular lipids apparently increase the number of ion-conducting defects around the enzyme, increasing Ca2+ efflux, and thereby reducing net uptake.