Lipopolysaccharide (LPS) endotoxins from Gram-negative bacteria have been extensively studied since their last review in 1990. LPS consists of a hydrophobic lipid A domain, a core oligosaccharide, and a distal polysaccharide (O-antigen). Recent genomic data have enabled detailed studies of LPS assembly in diverse Gram-negative bacteria, including human and plant pathogens. Key lipid A biosynthesis genes have also been found in higher plants, suggesting eukaryotic lipid A-like molecules may exist. The carbohydrate diversity of LPS is better understood, but much remains to be learned about its function. Gene comparisons suggest extensive lateral transfer of O-antigen assembly genes among bacteria. The most significant finding since 1990 is the identification of TLR4 as the lipid A signaling receptor in animal cells. TLR4 is part of a family of innate immunity receptors with a large extracellular domain, a single transmembrane segment, and a cytoplasmic signaling region that engages MyD88. Understanding TLR4 specificity and signaling pathways could provide new opportunities to block inflammatory effects of sepsis. Future progress will require insights into LPS-protein recognition at the atomic level, understanding of LPS trafficking, and biological approaches combining bacterial and animal genetics. Lipid A, the hydrophobic anchor of LPS, is a glucosamine-based phospholipid that makes up the outer monolayer of Gram-negative bacterial outer membranes. It is essential for bacterial growth and resistance to antibiotics and the complement system. Lipid A activates TLR4 in macrophages, triggering inflammation and co-stimulatory molecules for adaptive immunity. In mononuclear and endothelial cells, lipid A stimulates tissue factor production, aiding in infection clearance. However, overproduction in sepsis can damage blood vessels and cause septic shock. Synthetic E. coli lipid A causes similar effects when injected into animals, supporting its role in Gram-negative sepsis. The structural features of E. coli lipid A, especially its two phosphate groups and acyloxyacyl moieties, are needed for endotoxin response in human cells. The interaction of lipid A with animal cells has been elucidated, with lipid A delivered to CD14 on animal cell surfaces by a plasma lipid transfer protein. Recognition of lipid A by TLR4 is the earliest step in signal transduction. The interaction involves other proteins, including CD14 and MD2. Despite genetic evidence for TLR4 function, direct biochemical evidence for lipid A binding to TLR4 is still lacking. TLR receptors, including TLR4, function as dimers or heterodimers, similar to the IL-1 receptor. The three-dimensional structures of TLR extracellular domains are not yet determined, but the intracellular signaling domains of TLR1 and TLR2 have been reported.Lipopolysaccharide (LPS) endotoxins from Gram-negative bacteria have been extensively studied since their last review in 1990. LPS consists of a hydrophobic lipid A domain, a core oligosaccharide, and a distal polysaccharide (O-antigen). Recent genomic data have enabled detailed studies of LPS assembly in diverse Gram-negative bacteria, including human and plant pathogens. Key lipid A biosynthesis genes have also been found in higher plants, suggesting eukaryotic lipid A-like molecules may exist. The carbohydrate diversity of LPS is better understood, but much remains to be learned about its function. Gene comparisons suggest extensive lateral transfer of O-antigen assembly genes among bacteria. The most significant finding since 1990 is the identification of TLR4 as the lipid A signaling receptor in animal cells. TLR4 is part of a family of innate immunity receptors with a large extracellular domain, a single transmembrane segment, and a cytoplasmic signaling region that engages MyD88. Understanding TLR4 specificity and signaling pathways could provide new opportunities to block inflammatory effects of sepsis. Future progress will require insights into LPS-protein recognition at the atomic level, understanding of LPS trafficking, and biological approaches combining bacterial and animal genetics. Lipid A, the hydrophobic anchor of LPS, is a glucosamine-based phospholipid that makes up the outer monolayer of Gram-negative bacterial outer membranes. It is essential for bacterial growth and resistance to antibiotics and the complement system. Lipid A activates TLR4 in macrophages, triggering inflammation and co-stimulatory molecules for adaptive immunity. In mononuclear and endothelial cells, lipid A stimulates tissue factor production, aiding in infection clearance. However, overproduction in sepsis can damage blood vessels and cause septic shock. Synthetic E. coli lipid A causes similar effects when injected into animals, supporting its role in Gram-negative sepsis. The structural features of E. coli lipid A, especially its two phosphate groups and acyloxyacyl moieties, are needed for endotoxin response in human cells. The interaction of lipid A with animal cells has been elucidated, with lipid A delivered to CD14 on animal cell surfaces by a plasma lipid transfer protein. Recognition of lipid A by TLR4 is the earliest step in signal transduction. The interaction involves other proteins, including CD14 and MD2. Despite genetic evidence for TLR4 function, direct biochemical evidence for lipid A binding to TLR4 is still lacking. TLR receptors, including TLR4, function as dimers or heterodimers, similar to the IL-1 receptor. The three-dimensional structures of TLR extracellular domains are not yet determined, but the intracellular signaling domains of TLR1 and TLR2 have been reported.