The influence of cholesterol on the binding of alpha-crystallin to pure phospholipid membranes was studied. The rationale of this investigation stems from two unique aspects of human lens cells: an unusually high level of cholesterol in the membranes and the specific binding of alpha-crystallin to membranes. In the absence of cholesterol, binding of alpha-crystallin liposomes composed of either sphingomyelin, disteroyl-phosphatidylcholine or egg-phosphatidylcholine caused a decrease in the fluorescence intensity and anisotropy of the fluorophore NBD-PE. Since this fluorescence probe resides in the polar headgroup region of the membrane, the observed changes indicated that the binding of alpha-crystallin affected the structure of these membrane regions. The ability of alpha-crystallin to modulate membrane structure suggests yet another potential role for this lens protein. Addition of cholesterol markedly decreased the binding of alpha-crystallin to liposomes composed of either sphingomyelin or disteroylphosphatidylcholine and antagonized the capacity of bound alpha-crystallin to decrease membrane surface order. This antagonism could be explained by the ability of cholesterol to directly decrease the anisotropy of the fluorophore in sphingomyelin membranes unexposed to alpha-crystallin. Thus, with cholesterol present, a further decrease in membrane order upon subsequent binding of alpha-crystallin was less likely. The results obtained with the sphingomyelin liposomes are considered most meaningful, since sphingomyelins are the principal phospholipids in the human lens nuclear membrane and cholesterol preferentially interacts with sphingomyelin. We conclude that cholesterol in lipid membranes can antagonize the binding of alpha-crystallin and thus interfere with the capacity of bound alpha-crystallin to alter membrane order. We suggest that such actions of cholesterol might serve to preserve lens membrane structure in the physiological state where the concentration of soluble alpha-crystallin is great.