Mammalian cells respond to bacterial lipopolysaccharide (LPS) through a cognate receptor: Toll-like receptor 4 (TLR4). The signaling pathways, which link TLR4 to the proinflammatory transcription factor nuclear factor kappaB (NF-kappaB), occur through the intracellular docking proteins MyD88 and Trif. We hypothesize that unlike antigen-presenting cells, vascular endothelial cells (ECs) lack the Trif protein TRAM and are therefore incapable of eliciting Trif-dependent immune responses to LPS. Stimulation of wild-type mice with LPS leads to the activation of NF-kappaB in ECs and macrophages in vitro and in vivo. In contrast to macrophages, LPS did not activate endothelial NF-kappaB or NF-kappaB-dependent genes in MyD88(-/-) mice, suggesting the absence of a functional Trif pathway in vascular ECs. Indeed, the Trif-dependent gene cxcl10 was not expressed in ECs after LPS stimulation. This correlated with diminished expression of the Trif accessory TIR protein TRAM in ECs. Overexpression of TRAM cDNA in ECs reconstituted LPS-induced Trif-dependent NF-kappaB activation and cxcl10 promoter activity. The functional absence of TRAM in vascular ECs restricts TLR4 signaling to MyD88-dependent pathway. This is in contrast to macrophages, which respond to LPS via both Trif- and MyD88-dependent pathways. These findings indicate that vascular ECs do not express the Trif-dependent gene subset. This implies that these genes may be dispensable for the endothelial response to bacterial infection and play no role in the endothelial contribution to the development of atherosclerosis.