Xylitol inhibits the glycolysis and growth of Streptococcus mutans. We studied the inhibitory effect of xylitol on the acid production of S. mutans at several pH levels under the strictly anaerobic conditions found in the deep layer of dental plaque. Xylitol inhibited the rate of acid production from glucose and changed the profile of acidic end products to formate-acetate dominance, with a decrease in the intracellular level of fructose 1,6-bisphosphate and an intracellular accumulation of xylitol 5-phosphate (X5P). These results were notable at pH 5.5-7.0, but were not evident at pH 5.0. Since the activity of phosphoenolpyruvate phosphotransferase for xylitol was greater at higher pH, it is suggested that xylitol could be incorporated more efficiently at higher pH and that the resultant accumulation of X5P could inhibit the glycolysis of S. mutans more effectively.