The two acyl-homoserine lactones (AHLs) N-(butyryl)-L-homoserine lactone and N-[3-oxododecanoyl]-L-homoserine lactone (3-oxo-C₁₂-HSL) are required for quorum sensing in Pseudomonas aeruginosa. These AHLs derive their invariant lactone rings from S-adenosylmethionine and their variable acyl chains from the cellular acyl-acyl carrier protein (ACP) pool. This reaction is catalysed by specific AHL synthases, which exhibit acyl chain specificity. Culture supernatants of P. aeruginosa contain multiple 3-oxo-AHLs that differ in their acyl chain lengths but their physiological role, if any, remains unknown. An in vitro fatty acid-3-oxo-AHL synthesis system was established utilizing purified P. aeruginosa Fab proteins, ACP and the LasI 3-oxo-AHL synthase. In the presence of excess protein, substrates and cofactors, this system produced almost exclusively 3-oxo-C₁₂-HSL. When the β-ketoacyl-ACP reductase (FabG) catalysed step was made rate-limiting by switching from the preferred NADPH cofactor to NADH, increased levels of short chain 3-oxo-AHLs were produced, presumably because shorter-chain ketoacyl-ACPs accumulated and thus became LasI substrates. Consistent with these in vitro observations, a fabG(Ts) mutant produced increased amounts of 3-oxo-AHLs in vivo. Thus, in vitro and in vivo evidence indicated that modulation of FabG activity of the fatty acid biosynthetic pathway may determine the acyl chain lengths of these 3-oxo-AHLs and that the LasI 3-oxo-AHL synthase is sufficient for their synthesis.