Two crucial mediators of monocyte activation by lipopolysaccharide (LPS) are the acute phase plasma factor, lipopolysaccharide binding protein (LBP) and cell-surface-expressed CD14. Whether macrophage (M phi) recognized and respond to LPS in a similar manner is unknown. Here we show that human monocyte-derived M phi respond to LPS by tumor necrosis factor-alpha release and procoagulant activity upregulation by a similar dose response curve in the presence or absence of serum, suggesting that humoral factors such as LBP are relatively unimportant in the activation of M phi. Both serum-dependent and serum-independent activation of M phi by LPS require cellular CD14, as evidence by blocking studies with CD14-specific antibodies. Clones from the monocytoid cell line Mono Mac-6 selected for high LPS sensitivity displayed similar properties. When washed free of serum and cultured in the presence of calcitriol, they responded to LPS in a similar manner, regardless of the presence or absence of serum, and this response was inhibited by anti-CD14. It is hypothesized that during their differentiation. M phi acquire a functional substitute for the serum factor LBP, thereby being able to recognize low LPS concentrations in a milieu low in LBP concentration. It will be of interest to determine whether this is a high-affinity LBP receptor, LBP itself, or another cell surface constituent.