Muscle glucose uptake is greatly stimulated by moderate exercise, but full oxidation of the glucose to CO2 depends on the activity of the pyruvate dehydrogenase (PDH) complex. Our aim was to determine how PDH complex in different muscle groups responds to varying periods of moderate exercise. Rats were run on a motor-driven treadmill for 5-30 min and muscle PDH complex activity was determined in heart, diaphragm and red quadriceps muscles after isolation of mitochondria in the presence of inhibitors of PDH complex interconversion. In heart and diaphragm muscle, exercise caused an increase in PDH complex activity after 5 min, but this was followed by a significant decrease in activity as exercise progressed. In red quadriceps muscle, PDH complex activity was reduced after 5 min of exercise and was decreased further as exercise continued. We conclude that increased duration of exercise can lead to reduced PDH complex activity in rat muscles. We propose that this is a consequence of elevated fatty acid oxidation, the products of which stimulate PDH kinase. This implies that increased glycolysis to lactate and increased fatty acid oxidation can simultaneously provide energy for contracting muscle.