The intracellular pH (pH _i) optimum for glycolysis in Streptococcus mutans Ingbritt was determined to be 7.0 by use of the ionophore gramicidin for manipulation of pH_i. Glycolytic activity decreased to zero as the pH_i was lowered from 7.0 to 5.0. In contrast, glycolysis had an extracellular pH (pH_o) optimum of 6.0 with a much broader profile. The relative insensitivity of glycolysis to the lowering of pHo was attributed to the ability of S. mutans to maintain a transmembrane pH gradient (ApH, inside more alkaline) at low pH_o. At a pHo of 5.0, glycolyzing cells of S. mutans maintained a ΔpH of1.37 ± 0.09 units. The maintenance of thisΔpH was dependent on the concentration of potassium ions in the extracellular medium. Potassium was rapidly taken up by glycolyzing cells of S. mutans at a rate of 70 nmol/mg dry weight/min. This uptake was dependent on the presence of both ATP and a proton motive-force (Δp). The addition of N-N'dicyclohexylcarbodiimide (DCCD) to glycolyzing cells of S. mutans caused a partial collapse ofthe ΔpH. Growth of S. mutans at pH_o 5.5 in continuous culture resulted in the maintenance of a ApH larger than that produced by cells grown at pH _o 7.0. These results suggest the presence of a proton-translocating F₁F_o ATPase in S. mutans whose activity is regulated by the intracellular pH and transmembrane electrical potential (Δψ). The production of an artificial Δp of 124 mV across the cell membrane of S. mutans did not result in proton movement through the F ₁F_o-ATPase coupled to ATP synthesis. This suggests that ATP synthesis is not driven by the low Δp values obtained under physiological conditions in S. mutans. Therefore, the major role of the F₁ F_o-ATPase is likely to be in pH_i regulation. The extracellular concentration of sodium had no effect on ΔpH maintenance in glycolyzing cells, whereas the addition of 500 μmol/L fluoride caused a significant fall in pH_i. S. mutans was able to grow in the absence of a Δp, indicating that the transmembrane circulation of protons is not obligatory for growth of this organism.