In the present study, we used subcutaneous polyethylene glycol injections to show that a physiologically relevant stimulus, hypovolemia, will selectively increase the expression of neuropeptide genes in a restricted population of parvicellular corticotropin-releasing hormone-containing neurons in the hypothalamic paraventricular nucleus. Our results show that a large reduction in extracellular fluid maintained over approximately 20 hours is associated with a significant increase in the level of corticotropin-releasing hormone mRNA in the medial parvicellular division of the paraventricular nucleus. Additionally, there are concomitant increases in cellular levels of both neurotensin/neuromedin N and proenkephalin mRNAs. Our colocalization results show that the increases in neurotensin/neuromedin N and proenkephalin mRNAs after polyethylene glycol injection occur to a significant degree in cells that also contain corticotropin-releasing hormone mRNA. Furthermore, significant numbers of cells containing proenkephalin mRNA also contain neurotensin/neuromedin N mRNA, raising the possibility that some neurons have increased levels of all three mRNAs. Finally, in the medial parvicellular division of the paraventricular nucleus, the number of identified corticotropin-releasing hormone neurons also containing vasopressin mRNA is very low in control animals and is not increased by polyethylene glycol injections, suggesting that, within this period, activation of the vasopressin gene may not be a critical event in the neuroendocrine response of corticotropin-releasing hormone neurosecretory neurons to extracellular dehydration. Considered together with the effects of adrenalectomy on peptide colocalization, our results suggest the existence of several phenotypically distinct sets of neurons within the medial parvicellular division of the paraventricular nucleus, each characterized by its ability to regulate the expression of neuropeptide genes in a stimulus-specific manner.