Glucagon-like peptide-1 (GLP-1) and the GLP-1 receptor agonist, exendin-4 (Ex-4), potently stimulate hypothalamic-pituitary-adrenal (HPA) axis activity after either central or peripheral administration. Because several GLP-1 derivative drugs, including synthetic Ex-4, are currently in use to treat patients with type II diabetes mellitus, the characterization of Ex-4 effects on the HPA axis is highly relevant. Herein, the roles of CRH and AVP on these effects were investigated by administering the antagonists astressin and d(CH2)5Tyr(Me)AVP, respectively. The role of the sympathoadrenal system (SAS) was explored in bilateral adrenal enucleated and guanethidine-treated rats, whereas primary pituitary cell cultures were used to study direct effects on the corticotropes. Astressin completely abrogated (P < .05) the effects of Ex-4 central administration on ACTH secretion but only slightly reduced (by 35%) the ACTH response to Ex-4 peripheral administration. Moreover, astressin significantly (P < .05) decreased the corticosterone response to centrally but not peripherally administered Ex-4, suggesting different mechanisms depending on the route of administration. Pretreatment with d(CH2)5Tyr(Me)AVP failed to diminish either the ACTH or corticosterone response to Ex-4 and no direct effect of Ex-4 or GLP-1 was observed on pituitary cell cultures. In contrast, a significant (P < .05) reduction in the corticosterone response elicited by Ex-4 peripheral administration was observed in enucleated and guanethidine-treated rats, indicating a role of the SAS in the glucocorticoid stimulatory effects of Ex-4. Our data demonstrate that the effects of Ex-4 on the HPA axis are partially mediated by CRH and the sympathoadrenal system, and stress the relevance of Ex-4 as a corticosterone secretagogue.