In the hypothalamus, corticotropin-releasing factor (CRF) initiates the hypothalamic-pituitary-adrenal (HPA) axis response to stress, resulting in the release of glucocorticoids, including cortisol. Extrahypothalamic CRF, particularly in the limbic system, also appears to play a role in the stress response. To further define brain CRF response to stress, immunosensor-based microdialysis probes were used to measure the extracellular levels of CRF in the paraventricular nucleus of the hypothalamus (PVN) and in the amygdala of sheep during a predator (dog) exposure stress. In addition, gamma amino butyric acid (GABA) was measured in the amygdala and cortisol was measured in venous blood. Exposure to the predator stress increased CRF in the PVN and both CRF and GABA in the amygdala. These were followed in time by a rise in venous cortisol. Application of a CRF antagonist to the amygdala, immediately prior to stress, had a small effect on the subsequent observed stress responses. This treatment, however, significantly reduced the responses to a repeat stress administered 2 days later, compared to nontreated animals. Application of a GABA antagonist to the amygdala prior to stress had no effect on the subsequent observed stress response but increased the response to the stress repeated 2 days later. Perfusion with 4-aminopyridine, a neuronal depolarising agent, into the PVN induced a release of CRF accompanied shortly thereafter by a small increase in CRF in the amygdala, and 5-10 min later by an increase in venous cortisol. Perfusion into the amygdala increased the levels of both CRF and GABA but had no effect on either PVN CRF or venous cortisol. These data support roles for both the PVN and amygdala in stress responsiveness. It suggests further that actions at the amygdala can strongly influence subsequent responsiveness to a further stress, mediated in part by both CRF and GABA actions.