We have used colloid-induced hypovolemia to investigate mechanisms operating in CRH neuroendocrine neurons of the hypothalamic paraventricular nucleus during a sustained stress. Specifically, three questions have been addressed using in situ hybridization and RIA. 1) Do neuropeptide secretion and gene activation share the same stimulus threshold? 2) Does corticosterone modulate mechanisms regulating CRH gene expression during sustained stress? 3) How are neuropeptides commonly colocalized with CRH affected? Our results show that the secretion of ACTH and activation of the CRH gene have distinct and separate stimulus thresholds. The threshold is higher for CRH gene activation than for ACTH secretion, suggesting some degree of mechanistic separation. In addition, corticosterone secreted during the first 3 h of sustained hypovolemia does not inhibit CRH gene expression. However, feedback inhibition may occur in the delayed time domain. Finally, neuropeptides colocalized with CRH are differentially regulated by sustained hypovolemia. Proenkephalin messenger RNA levels show a slower temporal response than those of CRH, while the vasopressin gene is not activated at any time in parvicellular neuroendocrine neurons. Our results emphasize that CRH neuroendocrine neurons respond to a stress event in a stimulus-specific manner in terms of both the profiles of secretion and gene expression, and the structure of glucocorticoid feedback.