Many bacteria, including several pathogens of plants and humans, use a pheromone called an autoinducer to regulate gene expression in a cell density-dependent manner. Agrobacterium autoinducer [AAI, N-(3-oxo-octanoyl)-L-homoserine lactone] of A. tumefaciens is synthesized by the TraI protein, which is encoded by the tumor-inducing plasmid. Purified hexahistidinyl-TraI (H-6-TraI) used S-adenosylmethionine to make the homoserine lactone moiety of AAI, but did not use related compounds. H-6-TraI used 3-oxo-octanoyl-acyl carrier protein to make the 3-oxo-octanoyl moiety of AAI, but did not use 3-oxo-octanoyl-coenzyme A. These results demonstrate the enzymatic synthesis of an autoinducer through the use of purified substrates.