Pseudomonas aeruginosa uses quorum sensing to regulate gene expression in response to cell density. Two quorum sensing systems, las and rhl, use autoinducers—acyl-homoserine lactones—to activate transcriptional activators and control virulence genes. This study identifies a third signal, 2-heptyl-3-hydroxy-4-quinolone (PQS), which is a 4-quinolone that controls the expression of the lasB gene, which encodes the major virulence factor, LasB elastase. PQS is produced and bioactive through the las and rhl quorum sensing systems. The discovery of PQS expands the understanding of P. aeruginosa cell-to-cell signaling, showing that secondary metabolites like PQS can function as intercellular signals. PQS is not restricted to acyl-homoserine lactones and may play a role in regulating virulence gene expression. The study also highlights the potential of 4-quinolone signals as targets for antibacterial therapies. The identification of PQS provides insights into the complex quorum sensing hierarchy in P. aeruginosa and its role in virulence.Pseudomonas aeruginosa uses quorum sensing to regulate gene expression in response to cell density. Two quorum sensing systems, las and rhl, use autoinducers—acyl-homoserine lactones—to activate transcriptional activators and control virulence genes. This study identifies a third signal, 2-heptyl-3-hydroxy-4-quinolone (PQS), which is a 4-quinolone that controls the expression of the lasB gene, which encodes the major virulence factor, LasB elastase. PQS is produced and bioactive through the las and rhl quorum sensing systems. The discovery of PQS expands the understanding of P. aeruginosa cell-to-cell signaling, showing that secondary metabolites like PQS can function as intercellular signals. PQS is not restricted to acyl-homoserine lactones and may play a role in regulating virulence gene expression. The study also highlights the potential of 4-quinolone signals as targets for antibacterial therapies. The identification of PQS provides insights into the complex quorum sensing hierarchy in P. aeruginosa and its role in virulence.