We previously reported that the restriction of environmental space attenuated the hippocampal acetylcholine release and impaired spatial learning function. To examine the effect of the restriction of environmental space on the stress response of the hippocampal acetylcholine release, an in vivo microdialysis study was performed in male rats after 4 days of housing in a large cylindrical cage (diameter=35 cm) or a small cylindrical cage (diameter=19 cm). Significant stress response of the hippocampal acetylcholine release was observed in rats in the large cages (N=5), but it was not observed in rats in the small cages (N=5). The corticosterone concentration in serum was significantly increased by the restraint stress in both groups of rats. Although cage size does not influence stress-induced secretion of corticosterone, rats housed in a small cage exhibit lower levels of stress-induced ACh release than rats living in a large cage.