Xylitol inhibits the glycolysis and growth of Streptococcus mutans, but to different degrees among strains. Thus, we studied the biochemical mechanism through which the inhibition varies, using S. mutans strains ATCC 31989, NCTN 10449, and NCIB 11723, which are highly sensitive, moderately sensitive, and resistant to xylitol, respectively, under strictly anaerobic conditions such as those found in deep layers of dental plaque. Xylitol (30 mM) decreased the rate of acid production from glucose (10 mM) in ATCC 31989, NCTC 10449, and NCIB 11723 by 86, 26, and 0%, respectively. The activities of the xylitol: phosphoenolpyruvate phosphotransferase system (PEP-PTS) relative to those of glucose: PEP-PTS were 120, 16, and 3%, respectively. In ATCC 31989 and NCTC 10449, intracellular accumulation of xylitol 5-phosphate and decreases of fructose 1,6-bisphosphate and glucose 6-phosphate were observed. Furthermore, in the presence of xylitol (30 mM), glucose: PEP-PTS activities decreased by 34, 17, and 0%, respectively. These findings indicated that the higher the xylitol: PEP-PTS activity was and the more effectively xylitol decreased glucose: PEP-PTS activity, the more sensitive the strain was to xylitol. These results suggest that the following inhibitory mechanisms are active in the xylitol-sensitive mutans streptococci: direct inhibition of glycolytic enzymes by xylitol 5-phosphate derived from xylitol: PEP-PTS and, possibly, indirect inhibition through competition for the phosphoryl donor, HPr-P, between glucose and xylitol: PEP-PTSs.