Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-beta-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-beta-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-beta-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-alpha-l-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-alpha-l-arabinose in polymyxin-resistant strains of E. coli and Salmonella typhimurium.