The effect of spontaneous running exercise on muscle enlargement and sex differences in muscle hypertrophy were studied using morphological and biochemical approaches. Male and female weaning rats were placed in steel cages with free access to a running wheel for spontaneous exercise training. The running activity of female exercising rats reached more than twice the activity level of male exercising rats over 7 weeks of training. Muscle weights i. e., heart, gastrocnemius, and quadriceps, were increased in the male and female exercising groups; the quadriceps muscle weight increased with increasing running activity, and there was a significantly positive correlation between them. Morphological changes in muscle fibers were examined at the midpoint of the rectus femoris muscle. Male rats had much larger and longer muscle fibers than female rats, while there was no difference in fiber number between the sexes. Running exercise increased muscle fiber number and extended the fiber length significantly in both sexes, although the cross-sectional fiber area was not changed. Thus morphological studies at the midpoint of the muscle belly suggested that the spontaneous exercise-induced increase in muscle mass might be attributable to an increase in fiber number and extension of fiber length. Muscle RNA and DNA contents were increased in the male and female exercising groups. The amount of 3-methylhistidine excretion in urine was decreased in the female exercising group, but there was no change in the male groups. The RNA data supported the notion that spontaneous running exercise enhanced muscle protein synthesis in both sexes. It is concluded that the sex difference in the morphology of muscle fibers is present from the beginning, but that there is no sex difference in the direction of morphological or biochemical changes following spontaneous running exercise.