Overexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in the htrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). Since msbB displays 27.5% identity and 42.2% similarity to htrB, we explored the possibility that msbB encodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts of msbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB+ bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVA very rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype of htrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB.