Mu transposition recombination technique was firstly used as a mutagenesis tool to explore a cluster of genes involved in biosynthesis and regulation of pigments in P. aeruginosa. Eight pigment mutants were screened and identified. Gene cloning and sequencing of the region flanking the insertion revealed that the genes hmgA, ptsP, sucC, phzS, phzF1 were disrupted with mini-Mu respectively. Among them, gene hmgA is involved in the degradation of tyrosine, others affect the metabolism of pyocyanin which is the most important pigment of P. aeruginosa. Both gene phzS and gene phzF1 have been experimentally demonstrated to participate in pyocyanin synthesis. Gene ptsP belongs to a phosphotransferase system and gene sucC encodes succinyl-CoA synthetase beta chain. This is the first report that gene ptsP and sucC may be involved in the regulation of the biosynthesis of pyocyanin.