The inflammatory endothelial response to LPS is critical to the host surviving a gram-negative bacterial infection. In this study we investigated whether human endothelial cells express the functional co-receptor for LPS, CD14, and most importantly whether it is glycosyl-phosphatidyl-inositol (GPI) linked. We also examined whether plasma proteins could reconstitute an LPS response in CD14-inhibited endothelium. RT-PCR and CD14 specific MAbs demonstrated CD14 expression on primary Human umbilical vein endothelial cells (HUVEC) but not passaged HUVEC. The amino acid sequence of endothelial CD14 was 99% homologous to CD14 on monocytes. Endothelium responded to relatively low levels of LPS in the absence of plasma and this was entirely dependent on CD14. Removal of GPI linked proteins with Phosphatidyl inositol phospholipase C (PI-PLC) prevented LPS detection and subsequent protein synthesis (E-selectin expression). Endothelial CD14 was sufficient to initiate functional leukocyte recruitment, an event inhibited by blocking its LPS binding epitope and also by removing CD14 from the endothelial surface. Plasma proteins restored only about 30% of the LPS response in CD14-inhibited endothelium. In conclusion, our results strongly support an important role for endothelial membrane CD14 in the activation of endothelium for leukocyte recruitment.