This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype 06. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy-dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT, wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpL(O6) is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and A-band synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbp(O6) genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either 05 or 06; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.