We reported that gonadal steroid hormones sustain the stress-induced acetylcholine (ACh) release in the hippocampus in both sexes of rats. Since aromatase in the brain converts testosterone to estradiol, circulating testosterone activates both androgen and estrogen receptors in male rats. In the present study, to further determine the steroid hormone receptor which contributes to sustain the ACh release in male rats, an in vivo microdialysis study was performed after the letrozole (an aromatase inhibitor) or flutamide (an androgen receptor antagonist) treatment in male rats. Daily treatment with letrozole (5 mg/kg), flutamide (15 mg/kg), or vehicle was performed orally for 1 weeks. ACh concentrations in dialysates were assayed by the HPLC system, and the restraint stress was applied from 1200 h to 1300 h. Prior to the stress exposure, the ACh release was low in all male rats, and neither letrozole nor flutamide treatment showed significant effects on the basal ACh release. The ACh release in the hippocampus in male rats showed a prompt increase within 15 min after the onset of the restraint stress, but the increase in ACh release in letrozole- or flutamide- treated male rats was of a significantly smaller magnitude than vehicle-treated male rats. This decrease in the stress response was equivalent to that in gonadectomized rats. These results suggest that both androgen and estrogen receptors contribute to sustain the stress-induced acetylcholine release in the hippocampus in male rats. [Jpn J Physiol 55 Suppl:S183 (2005)]