luxSgene of Lactobacillus reuteri100-23C was amplified by PCR, cloned, and then sequenced. To define a physiological and ecological role for the luxSgene in L. reuteri100-23C, a luxSmutant was constructed by insertional mutagenesis. The luxSmutant did not produce autoinducers AI-2 or AI-3. Complementation of the luxSmutation by a plasmid construct containing luxSrestored AI-2 and AI-3 synthesis. In vitro experiments revealed that neither the growth rate, nor the cell yield, nor cell survival in the stationary phase were compromised in the luxSmutant relative to the wild type and complemented mutant. The ATP content of exponentially growing cells of the luxSmutant was, however, 65% of that of wild-type cells. Biofilms formed by the luxSmutant on plastic surfaces in a bioreactor were thicker than those formed by the wild type. Biofilm thickness was not restored to wild-type values by the addition of purified AI-2 to the culture medium. In vivo experiments, conducted with ex- Lactobacillus-free mice, showed that biofilms formed by the mutant strain on the epithelial surface of the forestomach were approximately twice as thick as those formed by the wild type. The ecological performance of the luxSmutant, when in competition with L. reuteristrain 100-93 in the mouse cecum, was reduced compared to that of a xylAmutant of 100-23C. These results demonstrate that LuxS influences important ecological attributes of L. reuteri100-23C, the consequences of which are niche specific.