The effect of cerulenin on conversion of an acyl-deficient precursor of lipid A to lipopolysaccharide was investigated in a mutant of Salmonella typhimurium (PRX22H9) conditionally defective in synthesis of 3-deoxy-D-mannooctulosonate (KDO). The precursor lacks both KDO and the saturated O-fatty acyl chains of lipopolysaccharide and contains beta-hydroxymyristate as sole fatty acid. Concentrations of cerulenin which inhibited de novo synthesis of fatty acids and lipopolysaccharide more than 95% had no effect on the rate or extent of conversion of preformed lipid A precursor to a lipopolysaccharide product. The product was identified as a polymer containing the Rc type core polysaccharide of PRX22H9 linked to the acyl-deficient lipid A unit of the precursor. The acyl-deficient lipopolysaccharide was translocated to the outer membrane at a normal rate. Lipopolysaccharide deficient in saturated fatty acids was also produced by a fabD mutant of Escherichia coli under conditions of limited endogenous fatty acid synthesis. The results indicate that prior incorporation of the saturated O-acyl chains of lipid A is not necessary for extension of the core polysaccharide chain and that synthesis of underacylated lipopolysaccharides occurs under conditions of restricted fatty acid synthesis which permit formation of the beta-hydroxymyristate-containing lipid A precursor.