Canine Purkinje fibers were isolated by microdissection and analyzed for four enzymes of glycogen metabolism and eight related metabolites. Purkinje fiber glycogen levels were very high, confirming earlier reports. Glycogen synthesizing enzymes, glycogen synthase and UDP glucose pyrophosphorylase, were on the average 47 and 70% higher, respectively, in Purkinje fibers than in myocardium. Phosphorylase activity was approximately equal in the two tissue types, and phosphoglucomutase was 31% lower in Purkinje fibers. The metabolites of glycogen 6-phosphate were all higher in Purkinje fibers (P less than 0.001), but glucose 1,6-bisphosphate was lower by 50%. Phosphocreatine and ATP remained high in Purkinje fibers during 2 min of ischemia, while the phosphocreatine level in myocardium was falling by 75%. The results of this study suggest that the high glycogen synthetic capability, high precursor levels, and overall lower metabolic rate in Purkinje fibers compared with myocardium may explain the much higher glycogen levels.