Phagocytosis of bacteria by monocytes and neutrophil granulocytes provides an important first line of defense against bacterial infections. Opsonization of bacteria with complement and phagocytosis by neutrophils is dependent on divalent cations and does not take place in blood that has been anticoagulated with EDTA. Monocytes, however, do carry out phagocytosis even in the presence of EDTA. We show here that this divalent cation-independent phagocytosis pathway requires the presence of the LPS receptor CD14 on the cell surface. This pathway is dependent on the availability of LPS binding protein, can be blocked by anti-CD14 Abs, by an excess of soluble CD14, by excess free LPS, or by an excess of unlabeled Gram-negative bacteria. In contrast, intact Gram-positive bacteria fail to inhibit this process. These experiments define a CD14-dependent phagocytosis pathway for Gram-negative bacteria that operates in monocytes in human whole blood. This pathway may be able to deal with bacterial pathogens that have developed resistance to complement-dependent opsonization and phagocytosis by neutrophils.