Trigger factor is a ribosome-associated peptidyl-prolyl cis/trans isomerase that is highly conserved in most bacteria. A gene, designated ropA, encoding an apparent trigger factor homologue, was identified in Streptococcus mutans, the primary etiological agent of human dental caries. Inactivation of ropA had no major impact on growth rate in planktonic cultures under the conditions tested, although the RopA-deficient mutant formed long chains in broth. Deficiency of RopA decreased tolerance to acid killing and to oxidative stresses induced by hydrogen peroxide and paraquat, and it reduced transformation efficiency about 200-fold. Addition of synthetic competence-stimulating peptide to the culture medium enhanced transformability of both the mutant and wild-type strains, although the ropA strain did not attain levels of competence observed for the parent. Loss of RopA decreased the capacity of S. mutans to form biofilms by over 80% when cultivated in glucose, but it increased biofilm formation by over 50% when sucrose was provided as the carbohydrate source. Western blot analysis revealed that the expression of glucosyltransferases B and D was lower in the RopA-deficient mutant. These results suggest that RopA is a key regulator of acid and oxidative stress tolerance, genetic competence, and biofilm formation, all critical virulence properties of S. mutans.