Quorum sensing (QS) is a bacterial cell-to-cell communication process that relies on the production, release, and response to extracellular signaling molecules called autoinducers. QS controls virulence and biofilm formation in the human pathogen Pseudomonas aeruginosa. P. aeruginosa possesses two canonical LuxI/R-type QS systems, LasI/R and RhlI/R, which produce and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Here, we use biofilm analyses, reporter assays, RNA-seq studies, and animal infection assays to show that RhlR directs both RhlI-dependent and RhlI-independent regulons. In the absence of RhlI, RhlR controls the expression of genes required for biofilm formation as well as genes encoding virulence factors. Consistent with these findings, ΔrhlR and ΔrhlI mutants have radically different biofilm phenotypes and the ΔrhlI mutant displays full virulence in animals whereas the ΔrhlR mutant is attenuated. The ΔrhlI mutant cell-free culture fluids contain an activity that stimulates RhlR-dependent gene expression. We propose a model in which RhlR responds to an alternative ligand, in addition to its canonical C4-homoserine lactone autoinducer. This alternate ligand promotes a RhlR-dependent transcriptional program in the absence of RhlI.