The cholesterol/phospholipid mole ratio (C/P) in the human erythrocyte membrane was varied by incubating cells with liposomes. The rotational mobility of band 3 proteins was measured in these membranes by observing flash-induced transient dichroism of the triplet probe eosin maleimide. Measurements were performed with membranes in which associations of band 3 with cytoskeletal proteins were removed by mild proteolysis with trypsin. It was found that decreasing C/P resulted in a more rapid decay of the flash-induced anisotropy. The anisotropy decay curves were analyzed by curve-fitting procedures, which indicated the existence of different sized small aggregates of band 3. The changes in the decay curves with varying C/P can be explained by an effect of cholesterol on the size distribution of these aggregates. The experiments suggest a possible role of cholesterol in regulating associations between integral membrane proteins.