Cultured cells possess high-affinity binding sites (receptors) for high density lipoprotein (HDL) that appear to mediate removal of excess intracellular cholesterol from cells. To examine the role of intact HDL apoproteins in receptor-mediated cholesterol removal, HDL3 apoproteins were digested with the proteolytic enzymes trypsin and pronase, and the residual particles were used in sterol efflux experiments. Protease treatment abolished the interaction of HDL3 with the 110-kd cell membrane protein postulated to represent the HDL receptor molecule, indicating that this interaction is mediated by HDL apoproteins rather than lipids. Compared with native HDL3 protease-modified HDL3 had a markedly reduced ability to selectively remove sterol from intracellular pools, even though modified particles promoted greater cholesterol efflux from the plasma membrane than did native particles. These results indicate that whereas sterol efflux from plasma membranes is mediated by HDL lipids, removal of excess intracellular sterol from cells is mediated by HDL apoproteins. These findings are consistent with the hypothesis that receptor binding of HDL apoproteins stimulates translocation of excess intracellular sterol to the cell surface where it becomes accessible for removal by HDL or other lipid-rich acceptor particles.