In isolated gastrocnemius muscles from 19 dogs the interstitial H+ activity ([H+]int) was measured with bulb-type buffer-filled glass minielectrodes. The muscles were working isotonically and perfused with blood. In addition arterial and venous pH, venous O2 saturation, muscle temperature, and blood flow were meausred continuously at rest, during 12 min of sustained exercise, and in the recovery period. Lactate (LA-) release and O2 consumption were calculated by the Fick principle. The experiments were performed under normal acid-base conditions and during artificially induced metabolic acidosis and alkalosis. 1. In normal acid-base balance [H+]int at rest was 54 +/- 3.3 neq/l (= pH 7.24), while venous H+ ([H+]ven) was 45 +/- 4.7 neq/l (= pH 7.34) A[H+] gradient was always observed between interstitial fluid and venous blood. 2. Immediately after onset of exercise [H+]int decreased transiently. After about 15 s [H+]int increased rapidly up to values of 105 +/- 7 neq/l (= pH 6.98). In the recovery period [H+]int diminished and reached control values after about 20-30 min. [H+]ven increased up to 74.4 +/- 8.4 neq/l (= pH 7.13). Maximal gradients between [H+]int and [H+]ven were 36 neq/l (= pH 0.2). 3. During repeated exercise the decrease in [H+]int at the onset of exercise was more extensive, while the subsequent increase was lowered. These changes correspond to a smaller LA- release. 4. During metabolic alkalosis at the onset of exercise [h+]int decreased less, during metabolic acidosis more than under normal acid-base conditions. Thereafter during metabolic alkalosis maximal values of 95.4 +/ 2 neq/l (= pH 7.03), during metabolic acidosis of 180 +/- 8.6 neq/l (= pH 6.74) were reached. This led to [H+] gradients between interstitial fluid and venous blood which were much higher in metabolic acidosis than in normal acid-base balance or in metabolic alkalosis. In metabolic acidosis [H+]int decreased very sle rapidly than during metabolic alkalosis or during normal acid-base conditions. It is concluded that the H+ activity measured is that within the interstitial space. Exercise hyperemia is not caused by changes of [H+]int. Mechanisms are discussed which may explain H+ gradients between interstitial fluid and venous blood and rapid changes of [H+]int at the onset of exercise.