Development of β-lactam resistance, production of alginate and modulation of virulence factor expression that alters host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. In this study, we propose that a co-regulatory network exists between these mechanisms. We compared the promoter activities of ampR, algT/U, lasR, lasI, rhlR, rhlI and lasA genes, representing the β-lactam antibiotic resistance master regulatory gene, the alginate switch operon, the las and rhl quorum-sensing (QS) genes, and the LasA staphylolytic protease, respectively. Four isogenic P. aeruginosa strains, the prototypic Alg(-) PAO1, Alg(-) PAOampR, the mucoid Alg(+) PAOmucA22 (Alg(+) PDO300) and Alg(+) PAOmucA22ampR (Alg(+) PDOampR) were used. We found that in the presence of AmpR regulator and β-lactam antibiotic, the extracytoplasmic function sigma factor AlgT/U positively regulated P(ampR), whereas AmpR negatively regulated P(algT/U). On the basis of this finding we suggest the presence of a negative feedback loop to limit algT/U expression. In addition, the functional AlgT/U caused a significant decrease in the expression of QS genes, whereas loss of ampR only resulted in increased P(lasI) and P(lasR) transcription. The upregulation of the las QS system is likely to be responsible for the increased lasA promoter and the LasA protease activities in Alg(-) PAOampR and Alg(+) PDOampR. The enhanced expression of virulence factors in the ampR strains correlated with a higher rate of Caenorhabditis elegans paralysis. Hence, this study shows that the loss of ampR results in increased virulence, and is indicative of the existence of a co-regulatory network between β-lactam resistance, alginate production, QS and virulence factor production, with AmpR playing a central role.