Three membrane properties were strikingly affected when the cholesterol of human erythrocytes, normally approximately 0.8 mol/mol of phospholipid (i.e. C/P approximately 0.8), was altered by equilibration with phospholipid dispersions of an appropriate cholesterol content. 1) While the sterol in intact red cell membranes of C/P less than or equal to 0.8 was resistant to cholesterol oxidase digestion, enrichment to C/P greater than or equal to 0.9 rendered the entire cholesterol pool sensitive to enzyme attack. Susceptibility to oxidation was reversed by removal of the excess cholesterol. Treatment of cells with 1 X 10(-4) M chlorpromazine also rendered the entire cholesterol pool susceptible to cholesterol oxidase. 2) Whereas ghosts with C/P less than or equal to 0.8 underwent invagination in low ionic strength, alkaline buffers to form inside-out vesicles, enrichment to C/P greater than or equal to 1.0 promoted right-side-out vesicle formation instead. This effect was mimicked by treatment with 2,4-dinitrophenol, an amphipath known to cause eversion of the membrane of red cells. In contrast, the presence of chlorpromazine, which promotes invagination of the intact red cell membrane, favored the formation of inside-out vesicles in ghosts. 3) The breakdown of ghosts into endocytic vesicles in dilute, alkaline media and the concomitant release and dissolution of the submembrane reticulum of spectrin and actin were retarded by excess cholesterol and promoted by its removal. This cholesterol effect was mimicked by exposing ghosts to chlorpromazine, but not dinitrophenol. Our data suggest that cholesterol may act physiologically both to stabilize the red cell membrane and to constrain its contour against invagination, and that red cell membrane cholesterol is maintained in vivo just below a critical level at which important organizational changes can occur.