4,5-Dihydroxy-2,3-pentanedione (DPD), a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). AI-2 is proposed to be a universal signal molecule mediating interspecies communication among bacteria. We show that mutualistic and abundant biofilm growth in flowing saliva of two human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by genetic or chemical (synthetic AI-2 in the form of DPD) complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Furthermore, an optimal concentration of DPD was determined, above and below which biofilm formation was suppressed. The optimal concentration was 100-fold lower than the detection limit of the currently accepted AI-2 assay. Thus, AI-2 acts as an interspecies signal and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity.