The LuxS protein is required for the biosynthesis of the type 2 autoinducer (AI-2), which is involved in quorum sensing in a wide range of bacterial species. We have determined the effects of a defined luxS mutation on the virulence of Streptococcus pneumoniae. Although the luxS mutant displayed reduced virulence relative to its wild-type parent, the type 2 strain D39, it was by no means avirulent in a mouse model. After intranasal administration, the luxS mutant was able to colonize the nasopharynx of the mouse as efficiently as the wild type. However, it was less able to spread from the nasopharynx to the lungs or the blood. Intraperitoneal coadministration studies indicated that the luxS mutant was less fit and was readily outcompeted by wild-type D39. However, when administered on its own by this route, the mutant was able to proliferate and cause fatal systemic disease, albeit at a lower rate than the wild type. Western blot analysis of whole-cell lysates of the mutant and its parent did not reveal any differences in the levels of several well-characterized virulence proteins. However, analysis of Coomassie blue-stained protein profiles after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that mutation of luxS had pleiotropic effects on protein expression in all cellular compartments. This is consistent with the product of luxS having a regulatory role in S. pneumoniae. This is the first report of a direct role for luxS (and by inference, AI-2) in the virulence of a gram-positive pathogen. However, the fact that mutagenesis of luxS does not completely attenuate S. pneumoniae has implications for the possible use of AI-2 antagonists for treatment of pneumococcal infections.