Skeletal muscle deaminates a substantial fraction of its adenylate pool to inosine 5'-monophosphate (IMP) when the rate of energy expenditure exceeds supply. How AMP deaminase is activated in vivo is unclear because the substrate affinity is quite low (Michaelis constant approximately 1-2 mM) relative to estimated concentrations of free AMP in skeletal muscle (0.2-1 microM). AMP deaminase:myosin binding causes a large increase in substrate affinity; whether this binding occurs during physiological exercise is uncertain. Exhaustive high-speed (60 m/min) treadmill exercise in rats results in an extensive depletion of adenine nucleotide and a stoichiometric accumulation of IMP (1.5-2 mumol/g) in the superficial vastus lateralis muscles (predominantly fast-twitch white). We measured AMP deaminase:myosin binding after intense exercise and found the bound fraction of AMP deaminase to be increased from 9 +/- 1% at rest to 48 +/- 4% at approximately 45 s after exercise. The extent of binding lessened during recovery from exercise, falling to 32 +/- 4% after approximately 75 s and 21 +/- 2% after approximately 105 s. This postexercise dissociation of AMP deaminase from myosin appeared to be a first-order process (approximately 50 s half time). Treadmill running that leads to deamination also results in AMP deaminase:myosin binding. Binding should activate AMP deaminase and thus favor IMP formation at low physiological concentrations of AMP.