Dynorphin (Dyn) peptides were previously shown to increase plasma corticotropin (ACTH) in the ovine fetus, but the site of its action remains unclear. In the present study, Dyn A1-17 was found to stimulate ACTH release from mouse anterior pituitary tumor AtT-20 cells in a dose-dependent manner. Naloxone did not block the effect of Dyn A1-17 and the selective ĸ-opioid receptor agonist U50488H did not stimulate ACTH release. Dyn A2-17, a degradative peptide fragment that does not bind to opioid receptors, also stimulated ACTH release from AtT-20 cells. Although the nonopioid effects of Dyn have previously been attributed to N-methyl-D-aspartate (NMDA) receptors, the ACTH-releasing effects of Dyn A1-17 in AtT-20 cells were not affected by co-administration of NMDA receptor antagonist LY235959. The ACTH response to Dyn A1-17 could not be blocked by α-helical CRH (CRH antagonist) and was additive with a maximal stimulatory dose of CRH, suggesting different mechanisms of action. These results show that the release of ACTH by Dyn A1-17 in AtT-20 cells is not mediated by ĸ-opioid receptors or by the NMDA receptor.