Maximal ADP-stimulated mitochondrial respiration depends on convergent electron flow through Complexes I+II to the Q-junction of the electron transport system (ETS). In most studies of respiratory control in mitochondrial preparations, however, respiration is limited artificially by supplying substrates for electron input through either Complex I or II. High-resolution respirometry with minimal amounts of tissue biopsy (1-3mg wet weight of permeabilized muscle fibres per assay) provides a routine approach for multiple substrate-uncoupler-inhibitor titrations. Under physiological conditions, maximal respiratory capacity is obtained with glutamate+malate+succinate, reconstituting the operation of the tricarboxylic acid cycle and preventing depletion of key metabolites from the mitochondrial matrix. In human skeletal muscle, conventional assays with pyruvate+malate or glutamate+malate yield submaximal oxygen fluxes at 0.50-0.75 of capacity of oxidative phosphorylation (OXPHOS). Best estimates of muscular OXPHOS capacity at 37 degrees C (pmol O(2)s(-1)mg(-1) wet weight) with isolated mitochondria or permeabilized fibres, suggest a range of 100-150 and up to 180 in healthy humans with normal body mass index and top endurance athletes, but reduction to 60-120 in overweight healthy adults with predominantly sedentary life style. The apparent ETS excess capacity (uncoupled respiration) over ADP-stimulated OXPHOS capacity is high in skeletal muscle of active and sedentary humans, but absent in mouse skeletal muscle. Such differences of mitochondrial quality in skeletal muscle are unexpected and cannot be explained at present. A comparative database of mitochondrial physiology may provide the key for understanding the functional implications of mitochondrial diversity from mouse to man, and evaluation of altered mitochondrial respiratory control patterns in health and disease.