The extracellular pH (pHo) and intracellular pH (pHi) were simultaneously measured with H(+)-sensitive microelectrodes in the rabbit papillary muscle during normal arterial perfusion and no-flow ischemia. The preparation was kept in an artificial gaseous atmosphere (N2 and CO2 during ischemia) without a surrounding fluid layer. Cylindrical muscles of small diameters (less than 1.0 mm) were selected to prevent major diffusion gradients of CO2 within the muscle cylinder during ischemia. In normal perfusion with CO2/HCO3(-)-buffered blood at PCO2 of 35 mm Hg, pHi was 7.03 +/- 0.03. During early ischemia, extracellular acidification was much more prominent than intracellular acidification. Consequently, the transmembrane pH gradient reversed (pHo less than pHi) at approximately 8 minutes. At 14 minutes of ischemia, pHo was 6.64 and pHi was 6.93. A moderate increase in PCO2 from 35 to 67 mm Hg before ischemia enhanced intracellular acidification in ischemia. Simulation of CO2 accumulation (increase of PCO2 in the surrounding atmosphere), as encountered in midmural ventricular layers during in vivo ischemia, produced a significant decrease of pHo (6.30 versus 6.64) and pHi (6.65 versus 6.93) at 14 minutes of ischemia. The presence of red blood cells in the intravascular space after arrest of coronary perfusion showed a pronounced effect on extracellular and intracellular acidosis. If the muscles were perfused with CO2/HCO3(-)-buffered perfusate in the absence of red blood cells, the changes of pHo and pHi were significantly larger (pHo, 6.00 versus 6.64; pHi, 6.46 versus 6.93 at 14 minutes) during ischemia. Actively developed force during ischemia was not significantly influenced by conditions modulating pHi. It decreased by 82% after 5 minutes, even when no significant change of pHi was recorded. By contrast, ischemic contracture was dependent on intracellular acidification. It developed earlier in the absence of red blood cells or with low extracellular buffer capacity. It is concluded that during acute myocardial ischemia 1) extracellular acidification exceeds intracellular acidification, 2) the decrease in pHi is inhomogeneous because of local variation in CO2 accumulation and diffusion, 3) the decrease in pHi is relatively small in the presence of red blood cells, and 4) the development of ischemic contracture but not the early decline in active tension is sensitive to changes in pHi.