Running becomes a stress, termed running stress, if it persists above the lactate threshold (LT) and results in enhanced plasma corticotropin (ACTH) level in humans. Although the exact underlying regulation mechanism is still uncertain, hypothalamic arginine-vasopressin (AVP) has been shown to play a dominant role in running-induced ACTH release. It is still not known, however, whether running stress activates the hypothalamic AVP-containing neurons that are involved in the activation of ACTH response. For this reason, we applied our rat running stress model, in which both plasma ACTH and osmolality levels increase just above the LT running (supra-LT running) in order to delineate which hypothalamic AVP neurons were responsive to running stress. Rats were previously habituated to running and then subjected to a 30-min run either just below or above the LT. Plasma samples were collected from these animals to determine ACTH and osmolality levels. Brains were prepared for immunocytochemistry for both AVP/Fos in the hypothalamus and enzyme immunoassay for the stalk median eminence (SME)-AVP content. Only supra-LT running resulted in an increase in the number of Fos/AVP immunoreactive neurons in both the parvocellular paraventricular nucleus (pPVN) and the magnocellular supraoptic nucleus (SON) accompanied by increased ACTH and plasma osmolality levels. Similarly, running reduced the SME content of the AVP. We thus found that AVP-containing neurons located in both the pPVN and SON are responsive to running stress just above the LT.