R ecognition of LPS by TLR4 on immune sentinel cells such as macrophages is thought to be key to the recruitment of neutrophils to sites of infection with Gram-negative bacteria. To explore whether endothelial TLR4 plays a role in this process, we engineered and imaged mice that expressed TLR4 exclusively on endothelium (known herein as Endothelium^TLR4 mice). Local administration of LPS into tissue induced comparable neutrophil recruitment in Endothelium^TLR4 and wild-type mice. Following systemic LPS or intraperitoneal E. coli administration, most neutrophils were sequestered in the lungs of wild-type mice and did not accumulate at primary sites of infection. In contrast, Endothelium^TLR4 mice showed reduced pulmonary capillary neutrophil sequestration over the first 24 hours; as a result, they mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wild-type mice in the first 48 hours. In fact, the only defect we detected in Endothelium^TLR4 mice was a failure to accumulate neutrophils in the lungs following intratracheal administration of LPS; this response required TLR4 on bone marrow–derived immune cells. Therefore, endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow–derived immune cells are critical for pathogen detection at barrier sites. Nonendothelial TLR4 contributes to failure to accumulate neutrophils at primary infection sites in a disseminated systemic infection.