Exposure of rat hearts perfused with 5 mM glucose and 5 mM acetate to tolbutamide led to a dramatic stimulation in glucose utilization and glycolytic flux. This effect was concentration dependent, with the largest response occurring at a tolbutamide concentration of 0.6 mM. Measurement of tissue glycogen content revealed that the higher concentrations of tolbutamide also enhanced glycogenolysis, indicating that the increase in glycolysis was caused by an increase in both glucose consumption and glycogen mobilization. On the basis of the determination of key metabolic intermediates, it was concluded that the stimulation of anaerobic metabolism was mediated by an activation of both phosphofructokinase and phosphorylase. The observed increase in glycolytic flux was associated with a rise in lactate production; however, the most pronounced effect of the drug was the stimulation of glucose oxidation. Thus the percentage of oxygen used in the oxidation of glucose was dramatically increased. Calculations also revealed that the contribution of glucose to overall ATP production rose from 8% in the absence of tolbutamide to about 30% in the presence of the sulfonylurea.