In a glucose-free bicarbonate Ringer (5% CO2 in N2), insulin increased intracellular pH (pHi), as determined by [14C]dimethadione, by 0.12 +/- 0.02 and stimulated glycolysis, as monitored by anaerobic lactate production, by 42.9 +/- 3.5% in paired frog sartorius muscles. The effect of insulin on glycolysis was shown to vary approximately linearly with log [Na+]0, being converted in 0.12 mM Na+ Ringer to a 51.5 +/- 8.4% inhibition of glycolysis. As the Na+ free-energy gradient was varied by decreasing [Na+]0 from 104 to 6.8 mM, the changes in glycolytic flux produced by insulin consistently paralleled the changes in pHi produced by the hormone. The relationship between the change in pHi and percent change in glycolytic flux was the same regardless of whether the effects were produced by insulin or by changing CO2. When glycolysis was either stimulated or inhibited, intracellular levels of fructose 6-phosphate varied inversely with glycolytic flux. This indicates that the effect on glycolysis of either insulin or changes in CO2 is due to a change in the activity of phosphofructokinase. The results support the model that the acute effect of insulin on glycolysis is mediated by a change in pHi, consequent to activation by insulin of Na:H exchange at the plasma membrane.