Septo-hippocampal cholinergic neurons play a significant role in the spatial learning and memory function. We found that corresponding sex steroid hormone is essential to maintain normal acetylcholine (ACh) release profile in the hippocampus of rats. In the present study, to further examine the short-term effect of sex hormones on the ACh release in the hippocampus, in vivo microdialysis study was performed in gonadectomized rats under the freely moving condition. The dialysate was collected from the dorsal hippocampus at 20-min intervals. Testosterone (2 mg) or 17β-estradiol (40 μg) was subcutaneously injected in gonadectmized male and female rats, respectively. The testosterone injection in gonadectomized male rats gradually but significantly increased the ACh release within the first few hours, and restored the ACh release in intact male rats within 10 hours. Similarly, the estrogen injection in gonadectomized female rats also increased the ACh release within the first few hours, and restored the ACh release in cycling female rats within 10 hours. Oil injection showed no significant effect on the ACh release. Present results demonstrated that testosterone in male rats as well as estrogen in female rats acutely increase the ACh release in the hippocampus. The short-term effect of gonadal steroid hormones may contribute to the enhancement of spatial mnemonic processing observed within the first few hours after the stroid treatemt. [J Physiol Sci. 2006;56 Suppl:S199]