A mutation in the msbB gene of Escherichia coli results in the synthesis of E. colilipopolysaccharide (LPS) that lacks the myristic acid moiety of lipid A. Although such mutant E. coli cells and their purified LPS have a greatly reduced ability to stimulate human immune cells, a minor reduction in the mouse inflammatory response is observed. When the msbB mutation is transferred into a clinical isolate ofE. coli, there is a significant loss in virulence, as assessed by lethality in BALB/c mice. When a cloned msbBgene is provided to functionally complement the msbBmutant, virulence returns, providing direct evidence that themsbB gene product is an important virulence factor in a murine model of E. coli pathogenicity. In the genetic background of the clinical E. coli isolate, themsbB mutation also results in filamentation of the cells at 37°C but not at 30°C, a reduction in the level of the K1 capsule, an increase in the level of complement C3 deposition, and an increase in both opsonic and nonopsonic phagocytosis of the msbBmutant, phenotypes that can help to explain the loss in virulence. The demonstration that the inhibition of msbB gene function reduces the virulence of E. coli in a mouse infection model warrants further investigation of the msbB gene product as a novel target for antibiotic therapy.