A pyocyanin overproducer with insertional inactivation of ptsP gene was isolated from a mini-Mu insertion library in Pseudomonas aeruginosa PA68. The mutation was complemented by a functional ptsP gene in trans. The pyocyanin-overproducing phenotype was also found in a ptsP mutant constructed by gene replacement in the P. aeruginosa PAO1 strain. Reporter plasmids with PqscR-lacZ, PlasI-lacZ and PrhlI-lacZ were constructed and the β-galactosidase activity in the ptsP mutant/wild-type background was measured. The results showed that lack of Enzyme INtr (EINtr, encoded by ptsP) decreased transcription from the PqscR promoter and increased the activity of the PlasI and PrhlI promoters. Normally, QscR represses the quorum-sensing LasR-LasI and RhlR-RhlI systems involved in pyocyanin regulation. Our results showed that the ptsP gene has an important role in the regulation of pyocyanin production and that two quorum-sensing systems and their repressor QscR are involved in this regulation.