The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.