Urocortin (Ucn) III, or stresscopin, is a high affinity ligand for the type 2 corticotropin-releasing factor (CRFR2) receptor recently identified in rodents and human. Ucn III was initially identified as a neuropeptide expressed in discrete areas in the brain. In the present study we demonstrate that Ucn III is expressed in pancreatic cells and a mouse -cell line, MIN6. Ucn III secretion from the cells was measured using a highly specific RIA and we found that high potassium, forskolin or high glucose can stimulate Ucn III secretion from these cells. In vivo studies showed that rats receiving an Ucn III injection intravenously had a significant elevation of plasma glucagon followed by plasma glucose levels compared with rats receiving vehicle. Ucn III injections also result in an increase in plasma insulin levels. The observed effects of Ucn III were blocked by pretreatment with a CRFR2 antagonist, astressin₂-B. Furthermore, Ucn III stimulated glucagon and insulin release from isolated rat islets and astressin₂-B abolished the effects of Ucn III, in keeping with a CRFR2 mediated mechanism. Taken together, the present studies suggest pancreatic Ucn III acting through CRFR2 is involved in the local regulation of glucagon and insulin secretion.