A. Eberhard,* AL Burlingame, C. Eberhard,* G. L. Kenyon, K. H. Nealson, and N. J. Oppenheimer abstract: Synthesis of bacterialluciferase in some strains of luminous bacteria requires a threshold concentration of an autoinducer synthesized by the bacteria and excreted into the medium. Autoinducer excreted by Photobacterium fischeri strain MJ-1 was isolated from the cell-free medium by ex-traction with ethylacetate, evaporation of solvent, workup with ethanol-water mixtures, and silica gel chromatography, fol-lowed by normal-phase and then reverse-phase high-perform-ance liquid chromatography. The final product was> 99% pure. The structure of the autoinducer as determined by high-resolution nuclear magnetic resonance spectroscopy, infrared spectroscopy, and high-resolution mass spectrometry was A-(3-oxohexanoyl)-3-aminodihydro-2 (3Ff)-furanone [or A-(/3-ketocaproyl) homoserine lactone]. The formation of homoserine by hydrolysis of the autoinducer was consistent with thisstructure. Synthetic autoinducer, obtained as a racemate, was prepared by coupling homoserine lactone to the ethylene glycol ketal of sodium 3-oxohexanoate, followed by mildly acidic removal of the protecting group; this synthetic material showed the appropriate biological activity.