Lipopolysaccharide (LPS), a major envelope component of Gram-negative bacteria, is the most frequent causative agent of septic shock and disseminated intravascular coagulation. LPS activates both CD14-positive (monocytes, macrophages, polymorphonuclear leukocytes) and CD14-negative (B-cell lines, endothelial cells) cells. CD14, a 55-kDa glycosyl-phosphatidylinositol-anchored membrane protein present on mature myeloid cells, serves as a receptor for LPS in complex with a soluble (serum-derived) LPS-binding protein (LBP). In this report, we show that human umbilical vein endothelial cells (HUVEC), which do not express measurable CD14 protein, become 3000-fold more sensitive to LPS-induced activation in the presence of serum, as measured by activation of the transcription factor NF-kappa B and expression of mRNA encoding tissue factor, a procoagulant molecule. This enhanced responsiveness of HUVEC is specifically mediated by the cell-free pool of CD14 (soluble CD14, sCD14) found in serum. The role of sCD14 in HUVEC activation by LPS was established by (i) the blocking effect of monoclonal anti-CD14 antibodies which discriminate between cell-bound and sCD14, (ii) the lack of the serum-enhancing effect after immunodepletion of sCD14, and (iii) establishing a reconstituted system in which recombinant sCD14 was sufficient to enhance the effects of LPS in the absence of serum and without a requirement for LBP. Thus, this mechanism of endothelial cell activation by LPS involves a cell-free pool of sCD14 most likely shed from CD14-positive cells of the monocytic lineage.