Acute nicotine is a potent stimulus for activation of the stress-responsive hypothalamic-pituitary-adrenal (HPA) axis, while chronic nicotine self-administration (SA) desensitizes the ACTH response to self-administered nicotine but cross-sensitizes to mild footshock stress (mFSS). To identify underlying mechanisms, we investigated (1) the effects of chronic nicotine SA on the coexpression of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) mRNAs, the primary hypothalamic neuropeptides regulating ACTH release, in the parvocellular division of paraventricular nucleus (pcPVN), and (2) mFSS-induced activation of these neurons during nicotine SA. Adult male Sprague Dawley rats were given 23 h/d unlimited access to self-administer nicotine (0.03 mg/kg per injection, i.v.) for 19 d. Brains were double labeled with fluorescence in situ hybridization of CRF and AVP mRNAs and triple labeled after mFSS exposure for CRF and AVP mRNAs and c-Fos protein. Chronic nicotine SA significantly increased AVP mRNA signal and the number of pcPVN AVP-positive (AVP(+)) neurons (twofold to threefold), reduced the number of CRF-positive (CRF(+)) neurons by approximately 60%, but increased pcPVN CRF(+)/AVP(+) neuronal number fivefold. Significantly, although chronic nicotine SA did not affect total c-Fos expression induced by mFSS in pcPVN CRF(+) neurons, the majority of the new CRF(+)/AVP(+) population was activated by this heterotypic stressor. These phenotypic neuronal alterations may provide the pivotal mechanism underlying the capacity of chronically self-administered nicotine to cross-sensitize the HPA response to specific stressors, suggesting that nicotine may augment HPA responsiveness to specific stressors in human smokers.