is a leading cause of life-threatening nosocomial infections. Many virulence factors produced by are controlled by the cell-to-cell communication process called quorum sensing (QS). QS depends on the synthesis, release, and groupwide response to extracellular signaling molecules called autoinducers. possesses two canonical LuxI/R-type QS systems, LasI/R and RhlI/R, that produce and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Previously, we discovered that RhlR regulates both RhlI-dependent and RhlI-independent regulons, and we proposed that an alternative ligand functions together with RhlR to control the target genes in the absence of RhlI. Here, we report the identification of an enzyme, PqsE, which is the alternative-ligand synthase. Using biofilm analyses, reporter assays, site-directed mutagenesis, protein biochemistry, and animal infection studies, we show that the PqsE-produced alternative ligand is the key autoinducer that promotes virulence gene expression. Thus, PqsE can be targeted for therapeutic intervention. Furthermore, this work shows that PqsE and RhlR function as a QS-autoinducer synthase-receptor pair that drives group behaviors in .