Oxidative slow skeletal muscle contains carbonic anhydrase III in high concentration, but its primary function remains unknown. To determine whether its lack handicaps energy metabolism and/or acid elimination, we measured the intracellular pH and energy phosphates by 31 P magnetic resonance spectroscopy in hind limb muscles of wild-type and CA III knockout mice during and after ischemia and intense exercise (electrical stimulation). Thirty minutes of ischemia caused phosphocreatine (PCr) to fall and Pi to rise while pH and ATP remained constant in both strains of mice. PCr and Pi kinetics during ischemia and recovery were not significantly different between the two genotypes. From this we conclude that under neutral pH conditions resting muscle anaerobic metabolism, the rate of the creatine kinase reaction, intracellular buffering of protons, and phosphorylation of creatine by mitochondrial oxygen metabolism are not influenced by the lack of CA III. Two minutes of intense stimulation of the mouse gastrocnemius caused PCr, ATP, and pH to fall and ADP and P i to rise, and these changes, with the exception of ATP, were all significantly larger in the CA III knockouts. The rate of return of pH and ADP to control values was the same in wild-type and mutant mice, but in the mutants PCr and Pi recovery were delayed in the first minute after stimulation. Because the tension decrease during fatigue is known to be the same in the two genotypes, we conclude that a lack of CA III impairs mitochondrial ATP synthesis.mitochondrial ATP synthesis S keletal muscle contains at least four isozymes of carbonic anhydrase (EC 4.2.1.1): CA II, CA III, CA IV, and CA V (1). CA II, the high-activity enzyme found in erythrocytes, is present in the sarcoplasm available to accelerate the removal of acid as CO 2 . CA IV is bound to the sarcolemma and facilitates lactate transport across the sarcolemma. CA V, in the mitochondria, accelerates the metabolism of pyruvate (2). CA III is present in the highest concentration of any of the carbonic anhydrases, as much as 2% of wet weight in slow oxidative muscle (type 1) (3), but its function is unknown. It is only minimally present in fast glycolytic (type 2) muscle (4). Changes in CA III levels have been reported when skeletal muscle is undergoing remodeling due to training (5) or inactivity (6, 7). CA III has a molecular weight of Ϸ30,000, similar to that of isozymes CA II, CA IV, and CA V, but accelerates CO 2 hydration at only 1/60th as much as human erythrocyte CA II and is 30,000 times less sensitive to acetazolamide (a widely used sulfonamide inhibitor) (8). It has two reactive surface cysteines, whereas the other muscle isozymes have none, indicating that it may have a role in the regulation of oxidative stress (9). In addition, CA III itself interacts directly with glutathione and is highly modified during lipid peroxidation (10, 11). Despite many experimental studies, no convincing rationale for the presence, preservation, and production of the large concentration of CA III in oxidative ...