To determine the significance of changes in cholesterol concentrations in various membranous components of liver cells, lean as well as genetically obese rats were fed diets containing cholesterol. Cholesterol was found to accumulate to a greater extent in the smooth vesicles (versus rough vesicles) of the endoplasmic reticulum, and the highest concentrations were in the plasma membrane fraction. These increases included a significant proportion of esterified cholesterol. Using 14C-mevalonate in vivo in rats fed a cholesterol-free diet, it was concluded (from the total activities of the isolated labeled cholesterol up to 180 minutes) that in time the total activity of newly synthesized cholesterol shifts from rough vesicles to smooth vesicles and finally to the plasma membrane fraction. However, 14C-cholesterol incorporation in vivo revealed a pattern of incorporation that was similar for smooth and rough vesicles in rats fed diets with or without cholesterol. Total activity of the rough was always higher. From the analytical data, esterification perhaps takes place at the level of the smooth vesicles (or cholesterol ester accumulation hastens the transition of rough to smooth). Polysomal profile analyses indicated that cholesterol feeding resulted in a relative decrease in larger aggregates in obese rats.