The incomplete lipid A precursor produced by a mutant conditionally defective in synthesis of 3-deoxy-D-mannoctulosonate (KDO) is rapidly converted to lipopolysaccharide when the mutant culture is shifted from nonpermissive to permissive conditions (Osborn, M.J., Rick, P.D., and Rasmussen, N.S. (1980) J. Biol. Chem. 255, 4246-4251). An intermediate product which accumulates transiently during this conversion has been isolated and identified as a derivative of the lipid A precursor containing 2 residues of KDO. The intermediate lacks the saturated O-fatty acyl chains of the completed lipid A and is indistinguishable in composition and chromatographic properties from the previously described product obtained by enzymatic addition of KDO to isolated lipid A precursor (Munson, R.S., Jr., Rasmussen, N.S., and Osborn, M.J. (1978) J. Biol. Chem. 253, 1503-1511). The intermediate produced in vivo is rapidly converted to lipopolysaccharide under conditions in which its continued formation is interrupted by return of the culture to nonpermissive temperature. The results provide direct evidence that transfer of KDO to lipid A precedes addition of the saturated fatty acid residues.