In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows. In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor-autoinducer pair. V. harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E. coli O157:H7, Salmonella typhimurium 14028, and S. typhimurium LT2 produce autoinducers similar or identical to the V. harveyi system 2 autoinducer AI-2. However, the domesticated laboratory strain E. coli DH5⣠does not produce this signal molecule. Here we report the identification and analysis of the gene responsible for AI-2 production in V. harveyi, S. typhimurium, and E. coli. The genes, which we have named luxS V.h. , luxS S.t. , and luxS E.c. respectively, are highly homologous to one another but not to any other identified gene. E. coli DH5⣠can be complemented to AI-2 production by the introduction of the luxS gene from V. harveyi or E. coli O157:H7. Analysis of the E. coli DH5⣠luxS E.c. gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxS E.c. protein. Our results indicate that the luxS genes define a new family of autoinducer-production genes.