To determine the effect of work rate on liver fructose 2,6-bisphosphate (fructose 2,6-P2), rats were run for 5 min on a treadmill up a 15% grade at 16, 21, 26, 31, and 36 m/min. The liver content of fructose 2,6-P2 decreased 25, 42, 50, 62, and 71% from resting values after 5 min of running at these work rates. The time course of the decline in liver fructose 2,6-P2 was also studied in rats run at 16 m/min for times ranging from 5 to 100 min, at 23 m/min for times ranging from 5 to 60 min, and at 31 m/min for times of 5, 10, and 20 min. The hepatic content of fructose 2,6-P2 declined significantly after 5 min in all three groups of rats. The rate of decline was greatest in rats run at 31 m/min. After 100 min of running, fructose 2,6-P2 in livers of rats running at 16 m/min declined to levels seen in rats run at 31 m/min for 20 min. Changes in fructose 2,6-P2 occurred before a detectable decline in liver glycogen and in the absence of any significant change in blood glucose. Liver adenosine 3',5'-cyclic monophosphate (cAMP) was elevated after 5 min of exercise in rats running at 23 and 31 m/min but not in rats running at 16 m/min. By the end of exercise, hepatic cAMP was elevated in rats running at all speeds. The rapid decline in fructose 2,6-P2 probably plays a role in decreasing hepatic glycolysis, thereby ensuring that glucose 6-phosphate derived from glycogenolysis is diverted to blood glucose.