To examine the sex difference in the 24-h profile of the acetylcholine (ACh) release in the hippocampus, in addition to the effects of housing conditions on this profile, we performed an in vivo microdialysis study in intact male and cycling female rats that had been living in large (diameter=35 cm) or small (diameter=19 cm) cylindrical cages. Each rat was individually housed in a cage for 4 days. On the day of the experiment, the dialysate was collected from the dorsal hippocampus at 20-min intervals and sequential blood samples were simultaneously obtained at 2-h intervals, under the freely moving condition for more than 24 h. ACh in the dialysates was measured by the high performance liquid chromatography system, while the corticosterone concentration in the serum was measured by radiostereoassay. Although the ACh release showed a clear daily rhythm in both sexes of rats, the amount of ACh released in female rats was significantly lower than that in males. Furthermore, the housing in the small cage significantly attenuated the ACh release during the dark phase in male rats, but not in female rats. Conversely, the serum corticosterone concentration showed a clear daily rhythm and the mean concentration of serum corticosterone in female rats was significantly higher than that in male rats. Housing in the small cage did not affect the corticosterone rhythm in either sex. These results reveal a sex difference in the 24-h profile of the ACh release, which suggests vulnerability of the cholinergic system in male rats depending on its housing conditions.