In vivo 31phosphorus nuclear magnetic resonance spectroscopy (P-NMR) of the anterior tibialis muscle was used to investigate the metabolic myopathy of inherited muscle-type phosphofructokinase (PFK) deficiency in four (homozygous) dogs who had mild exercise intolerance, rare muscle cramps, increased serum creatine kinase activity, but no myoglobinuria. During isometric muscle work induced by indirect electrical stimulation, and subsequent recovery, changes in the ratio of phosphocreatine (PCr) and inorganic phosphates (Pi) were comparable in muscle of PFK-deficient and normal dogs and indicated a large capacity for arobic oxidative phosphorylation in canine muscle. The progressive accumulation of sugar phosphates (PME) during graded exercise clearly demonstrated the glycolytic block in PFK-deficient dogs. During a muscle contracture, induced by acute muscle stimulation, PFK-deficient muscle became completely depleted of PCr and ATP, accumulated large amounts of PME, and recovered very slowly. We conclude that PFK-deficient dogs have a metabolic myopathy that demonstrated some but not all the features recognized in the human disorder.