Cholesterol was incorporated into Bacillus megaterium, resulting in reduction of membrane permeability and fluidity. However, an apparent modification of membrane composition was found neither in fatty acids nor in phospholipids, whereas electrophoretic patterns of the membrane proteins were changed. Unlike eukaryotic and mycoplasma cells, cholesterol incorporation into B. megaterium influences the membrane functions but is not accompanied by compensatory changes in the membrane lipids, which appear to be a response common to wall-covered and cholesterol-nonrequiring bacteria.