The concerted action of CRF and vasopressin (VP) plays a critical role in regulating ACTH release from anterior pituitary cells. In this study, we have explored the expression of these neurohormones in hypophysiotropic paraventricular neurons after repeated exposure of rats to immobilization stress. Cell by cell quantitative in situ hybridization was used to evaluate the steady state level of mRNAs coding for VP and CRF. We found that 16 daily stress exposures resulted in a significant increase in the average cellular level of CRF and VP mRNAs (150% and 200% of control levels, respectively). Moreover, in the repeatedly stressed group, the number of VP-expressing parvicellular neurons was approximately doubled relative to the control value. Using quantitative immunoelectron microscopy, VP- and CRF-immunoreactive sites were assessed in the dense core vesicle compartment of CRF axon terminals in the external zone of the median eminence. We found that after repeated stress, the immunolabeling of VP was augmented, while that of CRF was slightly decreased. Concurrently, we observed a significant increase in the proportion of CRF nerve terminals that were VP positive (from 50% in controls to 90% in stressed animals). We conclude that the observed changes in CRF neurons may represent a physiological response to increased functional demand and may lead to alterations in the composition of the ACTH-releasing signal.