The purpose of this study was to evaluate the relationship between mitochondrial Ca2+ concentration and the respiratory control index (RCI; state III/state IV) in isolated mitochondria before and after exhaustive exercise at 75% of maximal O2 consumption. Muscle biopsies of 100-150 mg from 12 moderately trained men were sampled at rest, immediately after exercise, and 30 or 60 min after exercise. The mitochondrial Ca2+ content after exhaustive exercise was significantly higher than the preexercise level [15.1 (range 39.4) vs. 11.6 (range 6.5) nmol/mg protein, respectively; P < 0.05], and RCI increased from 11.6 (range 14.4) at rest to 13.7 (range 15.0) at exhaustion (P < 0.05). After 60 min of recovery, the mitochondrial Ca2+ content was still high [18.8 (range 29.9) nmol/mg protein], but the RCI value was significantly depressed because of the increased state IV value and, in fact, was lower than the preexercise value [8.6 (range 5.1); P < 0.05]. Our results show that the mitochondrial Ca2+ content is increased in human skeletal muscle after prolonged exhaustive exercise and that this is followed by an elevated RCI value, with slightly increased state III and decreased state IV respiration. The restoration of the elevated mitochondrial Ca2+ level is slow and could be related to an increased state IV respiration, which together indicate uncoupled Ca2+ respiration during recovery.