To examine the role of gonadal steroid hormones in the stress-induced acetylcholine (ACh) release in the hippocampus, an in vivo microdialysis study was performed in gonadectomized (Gdx), gonadectomized and estrogen-primed (Gdx+E), and gonadectomized and testosterone-primed (Gdx+T) rats. Using both sexes of rats, the Gdx and steroid priming were performed at least 2 weeks before the experiment. ACh concentration in dialysates was assayed by the HPLC system, and the restraint stress was applied from 1200 h to 1300 h. In male rats, the ACh release in the hippocampus promptly increased within 15 min after the onset of the restraint stress in all groups of rats (P < 0.01 vs pre). Although the magnitude of ACh release response was low in Gdx or Gdx+E male rats, it was successfully ameliorated in Gdx+T male rats (P < 0.01). Also in female rats, the ACh release in the hippocampus promptly increased within 15 min after the onset of the restraint stress in all groups of rats (P < 0.01 vs pre). The magnitude of ACh release response was low in Gdx female rats, but was successfully ameliorated in Gdx+E and Gdx+T female rats (P < 0.05). In addition, Gdx+T male rats showed greater stress response than Gdx+E female rats (P < 0.05), that is indentical to the sex difference in gonadally intact rats. These results suggest that circulating testosterone in male rats as well as circulating estrogen in female rats contribute to sustain the stress response of ACh release in the hippocampus. [Jpn J Physiol 54 Suppl:S220 (2004)]