The expression of fructosyltransferase (FTF), the enzyme that synthesizes fructan from sucrose, is regulated in the cariogenic bacterium Streptococcus mutans. However, the exact mechanism of FTF regulation is unknown. In this study, the role of a two-component regulatory system (covRS) in FTF expression was investigated. A CovR-defective mutant of S. mutans NG8 was constructed by homologous recombination. By use of immunoblotting, the mutant was shown to overexpress FTF in the absence of sucrose, while the wild type and a covRS-complemented mutant showed sucrose-inducible FTF expression. Reverse transcription-PCR showed that the ftf transcript levels were increased in the covR mutant, suggesting regulation at the transcriptional level. The covR mutant was also found to overproduce extracellular carbohydrate, and this phenotype was reversed by covRS complementation. Paper chromatographic studies and chemical tests showed that the extracellular carbohydrate contained glucose and glucuronic acid but not fructose. These results suggest that the extracellular carbohydrate was not fructan. The production of a glucose- and glucuronic acid-containing extracellular carbohydrate has not been reported for S. mutans and may be considered novel. In conclusion, the results indicate that the expression of FTF and a glucose- and glucuronic acid-containing carbohydrate was negatively regulated by the covRS two-component regulatory system in S. mutans.