Despite the indication that the hypothalamo-pituitary-adrenal (HPA) axis is activated during treadmill running, there have not been any studies focusing on the relationship between exercise intensity and region-specific neural activities in hypothalamus. To address this, rats were subjected to 30 min of running, either at middle (supra-LT, 25 m min(-1)) or low speeds (sub-LT, 15 m min(-1)), and c-Fos-(+) cells were counted and compared with control rats. Significant increases in blood glucose and lactate levels, and plasma ACTH and osmolality levels were observed during supra-LT running. Only supra-LT running significantly increased c-Fos induction in various hypothalamic regions, namely, the medial preoptic area (MPO), periventricular nucleus (Pe), suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), parvocellular division of the paraventricular nucleus (pPVN), anterior hypothalamic area (AH), arcuate nucleus (ARC) and posterior hypothalamic nucleus (PH). However, sub-LT caused no effect on c-Fos accumulation. This indicates that the hypothalamus responds uniquely to running in a threshold-like pattern distinct from the speed-dependent pattern previously reported for the medulla oblongata [Ohiwa et al., 2006a,b]. In addition, these results showed a physiologic basis for mild exercise useful for establishing our minimum running stress (MRS) rat model, or the running conditions that minimize the activation of the HPA axis.