The biosynthesis of lipid A component was shown to be defective in a temperature-sensitive firA mutant of Escherichia coli. Cells were biosynthetically labelled with [14C]acetate and incorporation of radioactivity into the glycerophospholipid compared to lipid A fractions was measured. The lipid A/glycerophospholipid biosynthesis ratio of the firA mutant at 37 degrees C was approximately 50%, and at the nonpermissive temperature of 42 degrees C was less than 20% of that observed in the corresponding wild-type strain. Analysis of radiolabelled lipid A 4'-monophosphate derivatives and glycerophospholipids by thin-layer chromatography revealed that the firA mutant at 42 degrees C elaborated an altered lipid A, and its phosphatidylglycerol content was low. The chemical composition of the extracted lipopolysaccharides differed significantly between the firA and the wild-type strain only in the proportion of hexadecanoic acid, which was minimal in the wild type grown at 37 degrees C and 42 degrees C and in firA lipopolysaccharide grown at 37 degrees C. In the firA mutant lipopolysaccharide produced at 42 degrees C, hexadecanoic acid was present in approximately every third molecule, attached to the hydroxyl group of the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing glucosamine of lipid A. Inspection of dephosphorylated free lipid A preparations by laser-desorption mass spectrometry confirmed that significant amounts of heptaacyl lipid A was elaborated by the firA strain grown at 42 degrees C.