Isolated extensor digitorum longus muscles from rat were exposed to atmospheres of 30% CO2 (high-CO2 muscles) or 6.5% CO2 (control muscles) in O2 for 95 min. Muscle contraction characteristics were studied before and after the incubation. Tetanic tension decreased in high-CO2 muscles to 55% of initial value but remained unchanged in control muscles. Relaxation time was prolonged in high-CO2 muscles but not in control muscles. Intracellular pH was 6.67 +/- 0.04 (SD) in high-CO2 muscles and 7.01 +/- 0.04 in control muscles. CO2-induced acidosis had a marked influence on the intermediary energy metabolism as shown by a fourfold increase of glucose 6-phosphate, a 14% increase of ADP, and a decrease of phosphocreatine to 44% of the control value. Lactate and pyruvate contents were unchanged. The observed metabolic changes can be explained by an effect of H+ on the activity of phosphofructokinase and on the creatine kinase equilibrium. It can be concluded that H+ concentration causes muscular fatigue. It is, however, uncertain whether this is an effect of increased H+ per se or by high-energy phosphate depletion induced by acidosis.