Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme. Purified RhlI catalyzes the synthesis of acyl-HSLs from acyl–acyl carrier proteins and S-adenosylmethionine. An analysis of the patterns of product inhibition indicated that RhlI catalyzes signal synthesis by a sequential, ordered reaction mechanism in which S-adenosylmethionine binds to RhlI as the initial step in the enzymatic mechanism. Because pathogenic bacteria such as P. aeruginosa use acyl-HSL signals to regulate virulence genes, an understanding of the mechanism of signal synthesis and identification of inhibitors of signal synthesis has implications for development of quorum sensing-targeted antivirulence molecules.