Synthesis of the autoinducer signal involved in the cell density-dependent activation of Vibrio fischeri luminescence is directed by luxI. The autoinducer is N-(3-oxohexanoyl)homoserine lactone, and little is known about its synthesis. We have measured autoinducer synthesis by amino acid auxotrophs of Escherichia coli that contained luxI on a high-copy-number plasmid. Experiments with cell suspensions starved for methionine or homoserine show that either methionine or S-adenosylmethionine but not homoserine or homoserine lactone is required for autoinducer synthesis. The S-adenosylmethionine synthesis inhibitor cycloleucine blocks methionine-dependent autoinducer synthesis. Thus, it appears that S-adenosylmethionine rather than methionine is the molecule required for autoinducer synthesis. The amount of 15N-labeled methionine incorporated into the autoinducer by growing cultures of a homoserine and a methionine auxotroph was measured by mass spectrometry. The labeling studies show that even in the presence of homoserine, almost all of the autoinducer produced contains the 15N label from methionine. Thus, it appears that S-adenosylmethionine serves as the amino acid substrate in the luxI-dependent synthesis of the V. fischeri autoinducer.