Expression of the aminophospholipid phosphatidylserine (PS) on the surface of apoptotic lymphocytes and lipid-symmetric erythrocytes triggers their phagocytosis by macrophages. Phagocytosis by both activated and unactivated macrophages, which utilize different recognition systems, can be blocked by certain monoclonal antibodies directed against the LPS receptor, CD14. Here we investigate the requirement for CD14 in the phagocytosis of both apoptotic thymocytes and lipid-symmetric erythrocytes by both activated and unactivated macrophages. We show that phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages is completely abolished when CD14 is removed from macrophages by cleaving its glycosylphosphatidylinositol tether with phospholipase C. This treatment also substantially reduces phagocytosis of apoptotic lymphocytes by both types of macrophages. Unactivated LR-9 mouse macrophages which are deficient in CD14 expression are completely unable to phagocytose either apoptotic thymocytes or lipid-symmetric erythrocytes. These results argue that CD14 is an absolute requirement for the phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages, despite their different recognition systems, that CD14 contributes at least substantially to the phagocytosis of apoptotic lymphocytes by both activated and unactivated macrophages, and that activated macrophages may also possess an alternate, CD14-independent mechanism for phagocytosis of apoptotic lymphocytes.