Objective: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate production rates, which we refer to as the "MEGS capacity." Currently, little is known about MEGS pathology in patients with mutations in the mitochondrial DNA. Because MEGS capacity is an indicator for the overall mitochondrial function related to energy production, we searched for a correlation between MEGS capacity and 3243A→G mutation load in muscle of patients with the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) syndrome. Methods: In muscle tissue of 24 patients with the 3243A→G mutation, we investigated the MEGS capacity, the respiratory chain enzymatic activities, and the 3243A→G mutation load. To exclude coinciding mutations, we sequenced all 22 mitochondrial transfer RNA genes in the patients, if possible. Results: We found highly significant differences between patients and control subjects with respect to the MEGS capacity and complex I, III, and IV activities. MEGS-related measurements correlated considerably better with the mutation load than respiratory chain enzyme activities. We found no additional mutations in the mitochondrial transfer RNA genes of the patients. Interpretation: The results show that MEGS capacity has a greater sensitivity than respiratory chain enzymatic activities for detection of subtle mitochondrial dysfunction. This is important in the workup of patients with rare or new mitochondrial DNA mutations, and with low mutation loads. In these cases we suggest to determine the MEGS capacity. © 2008 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services.