Extracts of Escherichia coli contain an enzyme that generates the beta,1----6 linkage of lipid A from fatty-acylated monosaccharide precursors, according to the reaction: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2,3-diacyl-GlcN (beta, 1----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B. L., Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859). We now describe a membrane-bound kinase that phosphorylates the 4'-position of the above tetraacyldisaccharide 1-phosphate product. The lipid A 4'-kinase is distinct from the diglyceride kinase of E. coli. When crude membrane preparations are employed, several nucleoside triphosphates are able to support the phosphorylation of the tetraacyldisaccharide 1-phosphate, but ATP is the most efficient. The 4'-kinase requires Mg2+ and is stimulated by phospholipids, especially cardiolipin. Under optimal conditions the specific activity in crude extracts is 0.5 nmol/min/mg. The enzyme is rapidly inactivated by preincubation in the presence of detergents, such as Nonidet P-40 or octylglucoside, but phosphoenolpyruvate and glycerol stabilize the enzyme. The product generated in vitro has been characterized by fast atom bombardment mass spectrometry and by 1H and 31P NMR spectroscopy. Those analyses confirm that the 4' hydroxyl is the site of phosphorylation. The 4'-kinase reported here is likely to represent a key step in the de novo biosynthesis of lipid A.