We studied muscle biopsies of 103 pediatric patients in whom clinical suspicion for disorder of energy metabolism was highest in 13 patients, intermediate in 8 patients, and lowest in 82 patients. Electron transport complex (ETC) enzyme activity measurements were available in 96 of 103 patients. Most children with unclassified encephalopathy before biopsy had negative or equivocal morphological and biochemical evaluation for disorder of energy metabolism (72/85). The incidence of ETC abnormality and morphological abnormality in muscle from 39 patients with clinical encephalomyopathy (groups I, II, and III) was 20% and 38%, respectively. In 21 children with high or intermediate clinical suspicion of mitochondriopathy, light microscopy was confirmative in 12, ultrastructure was confirmative in 15, and major ETC abnormality was present in only 4 (29%) of 14. In 82 children with lower clinical suspicion of mitochondriopathy, morphological criteria at both the light and electron microscopic level were absent, and major abnormality of ETC activity was uncommon, in 9 (11%) of 82. Partial reductions of ETC activity occurred in 15 (18%) of 82, but are of uncertain significance. Ragged blue fibers were more prevalent in infants with mitochondriopathy than ragged red fibers. Increase of large, but not small, subsarcolemmal mitochondrial aggregates based on succinate dehydrogenase histochemistry is a useful indicator for mitochondriopathy. Thus, a distinction should be made between small aggregates (normal) and large aggregates. Using strict criteria to define pathological mitochondria, we concluded that electron microscopy is a powerful tool in the diagnosis of mitochondriopathy mainly when clinical suspicion is high. We found no consistent difference in the frequency of mitochondrial "proliferation" as currently defined or in citrate synthase activity in any group. Better patient selection in infants and children and better methods for investigation of mitochondriopathy are needed.