Pseudomonas aeruginosa is a common opportunistic pathogen and a model organism for studying bacterial sociality. A social behavior of P. aeruginosa that is critical for its success as a pathogen is its ability to form protective biofilms. Many of P. aeruginosa's social phenotypes are regulated by quorum sensing-a type of cell-cell communication that allows bacteria to respond to population density. Although biofilm formation is known to be affected by quorum sensing, evidence for direct regulation of biofilm production by quorum regulators has remained elusive. In this work, we show that production of the major biofilm matrix polysaccharide Psl in P. aeruginosa PAO1 is regulated by the quorum regulators LasR and RhlR in stationary-phase cultures. Secretion of Psl into the culture medium requires LasR, RhlR, and the quorum signal molecules N-3-oxo-dodecanoyl-homoserine lactone and N-butanoyl homoserine lactone. Psl production in strains unable to synthesize the homoserine lactone signals can be restored by exogenous introduction of the signal molecules. We found that LasR and RhlR perform different roles in the regulation of Psl production: LasR acts at the promoter of the psl operon and activates transcription of the Psl biosynthetic genes, while RhlR activates translation of the psl transcripts. This work contributes to our understanding of the overlapping but distinct functions of the Las and Rhl quorum-sensing systems and implicates both in the direct regulation of biofilm matrix production.IMPORTANCEPseudomonas aeruginosa biofilms are responsible for many treatment-resistant infections in humans. Many cooperative behaviors in P. aeruginosa are controlled by quorum sensing, but evidence for a direct role of quorum sensing in the regulation of biofilm matrix production has been scant. In this work, we show that the Las and Rhl quorum-sensing systems have distinct roles in regulating production of the matrix polysaccharide Psl and that this regulation happens at the level of transcription (Las) and translation (Rhl) of the psl operon. These findings deepen our understanding of overlapping functions of Las and Rhl quorum sensing and the complex regulation of biofilm development in P. aeruginosa.