Seven untrained volunteers [3 men, 4 women, 20.1 +/- 2.0 (SD) yr, 66. 0 +/- 11.0 kg, 171 +/- 13 cm] participated in a 10-day cycle exercise training program. Resting muscle samples were obtained from vastus lateralis before and after 5 and 10 days of training. Mitochondrial ATP production rate (MAPR) was assayed in isolated mitochondria by using a bioluminescence technique and referenced to the activity of glutamate dehydrogenase in the muscle sample. MAPR increased 136 and 161% after 10 days of training for the mitochondrial substrate combinations pyruvate + palmitoyl-L-carnitine + alpha-ketoglutarate + malate and palmitoyl-L-carnitine + malate, respectively. Total muscle glutamate dehydrogenase and citrate synthase activity increased 53 and 16%, respectively, after 5 days but did not significantly increase further after 10 days. The results from the present study indicate that MAPR, measured by using the substrate combinations pyruvate + palmitoyl-L-carnitine + alpha-ketoglutarate + malate and palmitoyl-L-carnitine + malate, can rapidly increase in response to endurance training.