To determine whether severe long-term exercise affects on the brain, we investigated the mice brain after 12-week treadmill exercise. The mice (ddN, male, 25-35 g in body weight) were divided into severe, mild, and non-exercise group. Mice in severe groups ran on a treadmill at a speed of 25 m/min for 12 weeks and mice in mild group ran on a treadmill at a speed of 10 m/min for 12 weeks. The mice were killed by transcardial perfusion with 0.1 M phosphate-buffered saline (PBS) followed by ice-cold 4% paraformaldehyde in 0.1 M PB. The another sets of mice were fixed by 2.5% glutaraldehyde-2% osmium tetroxide for electromicroscope (EM). The brains were serially sectioned in the coronal plane at a thickness of 20-microns with a vibratome and then processed for histology, by means of hematoxylin-eosin (HE) staining and immunohistochemistry. Fifty % of mice in severe exercise showed hyperchromatic and shrunken nerve cells with nuclear pyknosis (dark neuron) in the hippocampus, but not in mild exercise and non-exercise groups. The immunoreactivity of microtuble associated protein-2 (MAP-2) decreased, while the heat-shock protein/cognate 70 (HSP/C 70) increased in the hippocampus of severe exercise group. Many destroyed mitochondria were observed in dark neurons by Electron micrograph. These findings suggested that severe long-term exercise might damage hippocampal neurons.