An endogenous adrenergic antagonist extracted from porcine duodenal mucosa was tested for 3H-yohimbine (5 nM) binding inhibitory activity using whole brain membranes. Duodenal mucosa was scraped into liquid N2 and extracted in 2M acetic acid (HAc) + 1 mg/ml ascorbic acid. The supernatant was fractionated on SP-Sephadex C-25 with a stepwise pH gradient. The peak adrenoceptor binding inhibitory (ABI) activity eluted at pH 4.5 and fractionation of this material on Sephadex G-25 SF indicated a relative molecular mass (Mr) of 2000 to 3000. The ABI did not bind to a Pharmacia Pro RPC 5/5 column which resulted in further purification and indicated that the peptide is hydrophilic. ABI activity was specific for 3H-yohimbine and did not affect 3H-dihydroalprenolol binding to beta receptors. Incubation of pancreatic islets isolated from fasted rats with ABI completely reversed the effect of 2.5 X 10(-6)M norepinephrine (NE) on insulin release in the presence of 300 mg/dl glucose. I.v. injection of ABI in rats also prevented the increased serum glucose response to a glucose bolus caused by NE. Preliminary experiments indicate that ABI blocks the stimulation of platelet aggregation by epinephrine as well. The peptide identified in this study causes specific inhibition of binding to alpha-2 adrenergic receptors and exerts a potent glucose dependent effect on adrenergic inhibition of insulin release. Possible implications in diabetes mellitus are discussed.