The purpose of ihese experiments was to obtain information, under controlled conditions, regarding the effect of regularly performed exercise on the concentration of myoglobin in skeletal muscle. Rats were subjected to a vigorous program of treadmill running of progressively increasing intensity lasting 15 weeks. This program resulted in a highly significant increase in the capacity for prolonged running. The weight and water content of the leg muscles was unaffected. The myoglobin concentration in the quadriceps and hamstring muscles increased approximately 80%, while that of the abdominal muscles was unaffected. This suggests that a local factor, limited to the exercising muscles, was responsible for the increase. The level of habitual physical activity may play an important role in determining the concentration of myoglobin in skeletal muscle.