Onset of intracellular acidosis during muscular exercise has been generally attributed to activation or hyperactivation of nonoxidative ATP production but has not been analyzed quantitatively in terms of H⁺ balance, i.e., production and removal mechanisms. To address this issue, we have analyzed the relation of intracellular acidosis to H⁺ balance during exercise bouts in seven healthy subjects. Each subject performed a 6-min ramp rhythmic exercise (finger flexions) at low frequency (LF, 0.47 Hz), leading to slight acidosis, and at high frequency (HF, 0.85 Hz), inducing a larger acidosis. Metabolic changes were recorded using ³¹P-magnetic resonance spectroscopy. Onset of intracellular acidosis was statistically identified after 3 and 4 min of exercise for HF and LF protocols, respectively. A detailed investigation of H⁺ balance indicated that, for both protocols, nonoxidative ATP production preceded a change in pH. For HF and LF protocols, H⁺ consumption through the creatine kinase equilibrium was constant in the face of increasing H⁺ generation and efflux. For both protocols, changes in pH were not recorded as long as sources and sinks for H⁺ approximately balanced. In contrast, a significant acidosis occurred after 4 min of LF exercise and 3 min of HF exercise, whereas the rise in H⁺ generation exceeded the rise in H⁺ efflux at a nearly constant H⁺ uptake associated with phosphocreatine breakdown. We have clearly demonstrated that intracellular acidosis in exercising muscle does not occur exclusively as a result of nonoxidative ATP production but, rather, reflects changes in overall H⁺ balance.