We studied the mechanism by which the antimicrobial compound sucrose monolaurate inhibits Streptococcus mutans NCTC 10449 by determining its effect on the rate of acid production from glucose and sucrose and the intracellular and extracellular levels of glycolytic intermediates. Sucrose monolaurate was more effective than either sodium laurate or sodium fluoride in inhibiting acid production at pH 7.0 from glucose. Inhibition of acid production was the same when either glucose or sucrose was the carbon source and in the presence or absence of oxygen. Quantitative analysis of various glycolytic intermediates revealed that the steps inhibited by sucrose monolaurate were the reactions catalyzed by phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and/or phosphoglycerate kinase. Since the activities of these enzymes in cell-free extracts were not decreased by the addition of sucrose monolaurate, the inhibition of acid production could not be ascribed to direct effects on the enzymes. A decrease in the rate of acid production with corresponding elevations in the extracellular levels of glycolytic intermediates indicates that sucrose monolaurate inhibits S. mutans by altering the permeability of the cell membrane, which causes a loss of important metabolites.