L-Rhamnose (L-Rha) is a component of the lipopolysaccharide (LPS) core, several O antigen polysaccharides, and the cell surface surfactant rhamnolipid of Pseudomonas aeruginosa. In this study, four contiguous genes (rmlBDAC) responsible for the synthesis of dTDP-L-Rha in P. aeruginosa have been cloned and characterized. Non-polar chromosomal rmlC mutants were generated in P. aeruginosa strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extracted from the mutants was analysed by SDS-PAGE and Western immunoblotting. rmlC mutants of both serotype O5 and serotype O6 synthesized a truncated core region which was unable to act as an attachment point for either A-band or B-band O antigen. A rmd rmlC PAO1 double mutant (deficient in biosynthesis of both D-Rha and L-Rha) was constructed to facilitate structural analysis of the mutant core region. This strain has an incomplete core oligosaccharide region and does not produce A-band O antigen. These results provide the genetic and structural evidence that L-Rha is the receptor on the P. aeruginosa LPS core for the attachment of O polysaccharides. This is the first report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of P. aeruginosa LPS, and provides further insight into the mechanisms of LPS biosynthesis and assembly in this bacterium.