The present study investigated the effects of intensity of treadmill exercise and duration of mild-treadmill exercise on activity of 5-HT in the cerebral cortex and dentate gyrus of rats via immunohistochemistry. For intensity-dependence treadmill exercise, rats were randomly assigned to four groups: the control group, the mild-exercise group, the moderate-exercise group, and the severe-exercise group (n = 10 in each group). For duration of mild-treadmill exercise. animals were divided into six groups: the control group, the 1 day-exercise group, the 3 days-exercise group, the 1 week-exercise group, the 2 weeks-exercise group, the 4 weeks-exercise group (n = 10 in each group). Thirty minutes after each exercise, rat brains were surgically removed for the histochemical determination of the 5-HT activity. The results were as follows; 1. 5-HT activity of the cerebral cortex was increased from treadmill exercise with increment of intensity. but no significant difference observed between the control group and the mild-exercise group. However, 5-HT activity of the dentate gyrus was increased in the mild-exercise group, and which it decreased with increasing of intensity. Also, no significant difference observed between the control group and the severe-exercise group. 2. In duration of mild-treadmill exercise. 5-HT activity of the cerebral cortex was increased the maximum level in the ] week of exercise, after which it gradually decreased. However, 5-HT activity of the dentate gyrus reached the maximum level on the 3 days of exercise and it maintained until 2 weeks, which it gradually reduced. Overall, there are different tendency represented impact of intensity of treadmill exercise on the 5-HT synthesis between cerebral cortex and dentate gyrus that is involved in the affect and the learning and memory. These results indicate that although the exercise can evoke central fatigue, whereas it could give a positive effect on learning and memory. and it suggests that the mild-exercise is more effective in terms of increasing learning and memory.