The energetics of human muscle have been investigated in vivo during and after fatiguing aerobic, dynamic exercise. Changes in cytoplasmic pH and concentrations of phosphocreatine, ATP and Pi were followed using 31P nuclear magnetic resonance spectroscopy. ATP was significantly depleted in 6 out of 12 experiments and in these 6 experiments decreased to 55 +/- 5% of the pre-exercise concentration. Depleted muscle had a lower phosphocreatine concentration (17 +/- 5% of resting value) and lower pH (6.12 +/- 0.04) than fatigued muscle in which ATP loss was not observed (26 +/- 5% for phosphocreatine and 6.37 +/- 0.09 for pH). The free energy of hydrolysis of ATP was not significantly different in the two groups and was also similar in exhausted and nonexhausted muscle. Loss of ATP was associated with altered recovery of the muscle: [phosphocreatine], [Pi], and pH returned more slowly to their pre-exercise values and the initial rate of oxidative phosphorylation was diminished. The restitution of [ATP] to its pre-exercise value was much slower than that of the other metabolites.