The relative contribution of O2 and CO2 to the metabolic control of blood flow in long-term exercise was examined in the denervated gracilis muscle of the anesthetized dog. The data show that 1) on initiation of heavy exercise, the effluent blood PO2 and pH fall markedly and then rise slowly but remain depressed relative to control during 60 min of exercise hyperemia, while the initial increases in [K+] and osmolality rapidly approach and eventually reach preexercise levels. 2) The enhanced vasodilator activity of venous blood from exercising muscle is attenuated when effluent blood PO2 or pH is corrected to preexercise levels; it is completely abolished when both are corrected. 3) Induced reduction PO2 or pH in the arterial inflow, and thus venous outflow, of resting muscle produces a fall in resistance; simultaneous reductions of both to levels seen in heavy exercise produce a fall in resistance to near that observed during exercise. Since the enhanced vasodilator activity of venous blood from the contracting muscle was abolished by simultaneous correction of the PO2 and pH, it seems likely that these factors, acting directly or indirectly, are the principal chemicals responsible for the maintenance of the vasodilation seen in canine skeletal muscle during heavy exercise.