Abstract – It has been reported previously that xylitol added to glucose used to challenge dental plaque in vivo caused a reduced acid formation. The aim of the present study was to approach the mechanism by which xylitol may affect glucose catabolism in plaque bacteria. Suspensions of freshly collected 4‐day‐old plaque bacteria were ineubated, one batch with labeled xylitol, one with labeled glucose, in vitro at 37°C. Samples of cells were taken out at time intervals, collected on paper discs and subjected to scintillation counting. It was observed that the plaque bacteria took up xylitol, the uptake increasing with incubation of more than 3–4 h, whereas the same cells took up glucose immediately. Cells which had taken up xylitol were extracted with boiling water, extracts concentrated and applied on thin‐layer chromatography sheets. A radioactive component with mobility like xylitol‐5‐phosphate was isolated from the cell extracts, and also a component where labeled xylitol was associated with macromolecules. It is suggested that the accumulation of the metabolities within the cells inhibits glycolysis.