TLR4 is a member of the Toll-like receptor family and has been identified as the gene necessary for potent responses to lipopolysaccharide (LPS) in mammals. This study demonstrates that TLR4 is involved in LPS-induced activation of the nuclear factor-κB (NF-κB) pathway. HEK 293 cells transiently transfected with human TLR4 cDNA and an NF-κB-dependent luciferase reporter plasmid showed that LPS stimulates NF-κB-mediated gene expression in a dose- and time-dependent manner. The LPS antagonist E5531 blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50 ~30 nM), indicating that TLR4 serves as a cell-surface co-receptor for CD14, leading to LPS-mediated NF-κB activation. LPS, a component of Gram-negative bacteria, is a potent activator of mammalian cells and is involved in the development of Gram-negative sepsis. LPS activates cells through interaction with CD14, a cell surface glycoprotein. TLR4 is a transmembrane receptor that, when activated by LPS, leads to NF-κB activation and subsequent inflammatory responses. TLR4 is highly expressed in cells that respond to LPS, such as macrophages and monocytes. The C3H/HeJ mouse, which is resistant to LPS, has a mutation in the TLR4 gene, supporting the idea that TLR4 is the dominant LPS receptor in mammals. The study shows that TLR4 is involved in LPS signaling and serves as a cell-surface co-receptor for CD14. E5531, an LPS antagonist, inhibits TLR4-mediated NF-κB activation in a dose-dependent manner, indicating that TLR4 is a receptor for LPS. These findings support the hypothesis that TLR4 is essential in LPS signaling in vitro and in vivo. The results also highlight the importance of TLR4 in the development of anti-endotoxin agents.TLR4 is a member of the Toll-like receptor family and has been identified as the gene necessary for potent responses to lipopolysaccharide (LPS) in mammals. This study demonstrates that TLR4 is involved in LPS-induced activation of the nuclear factor-κB (NF-κB) pathway. HEK 293 cells transiently transfected with human TLR4 cDNA and an NF-κB-dependent luciferase reporter plasmid showed that LPS stimulates NF-κB-mediated gene expression in a dose- and time-dependent manner. The LPS antagonist E5531 blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50 ~30 nM), indicating that TLR4 serves as a cell-surface co-receptor for CD14, leading to LPS-mediated NF-κB activation. LPS, a component of Gram-negative bacteria, is a potent activator of mammalian cells and is involved in the development of Gram-negative sepsis. LPS activates cells through interaction with CD14, a cell surface glycoprotein. TLR4 is a transmembrane receptor that, when activated by LPS, leads to NF-κB activation and subsequent inflammatory responses. TLR4 is highly expressed in cells that respond to LPS, such as macrophages and monocytes. The C3H/HeJ mouse, which is resistant to LPS, has a mutation in the TLR4 gene, supporting the idea that TLR4 is the dominant LPS receptor in mammals. The study shows that TLR4 is involved in LPS signaling and serves as a cell-surface co-receptor for CD14. E5531, an LPS antagonist, inhibits TLR4-mediated NF-κB activation in a dose-dependent manner, indicating that TLR4 is a receptor for LPS. These findings support the hypothesis that TLR4 is essential in LPS signaling in vitro and in vivo. The results also highlight the importance of TLR4 in the development of anti-endotoxin agents.