The functional role of opioid receptors in acetylcholine release from splanchnic nerve terminals in the adrenal medulla was investigated in halothane-anesthetized rats. The extracellular acetylcholine level was measured by a newly developed in vivo adrenal microdialysis method. The potassium (K+)-evoked acetylcholine release from the splanchnic nerve terminals was inhibited by morphine (10 microM), a mu-opioid receptor agonist, and [D-Pen2,D-Pen5]enkephalin (DPDPE, 1 and 10 microM), a delta-opioid receptor agonist. These inhibitory effects of morphine and DPDPE were significantly abolished by naltrexone (9 mg/kg i.p.), a mu-opioid receptor antagonist, and naltrindole (9 mg/kg i.p.), a delta-opioid receptor antagonist, respectively. 5 alpha,7 alpha-beta-(-)- N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]benzene acetamide (U69593, 10 microM), a kappa-opioid receptor agonist, had no influence on the K(+)-evoked acetylcholine release. The findings suggest that both mu- and delta-opioid receptors might have a functional role in acetylcholine release from splanchnic nerve terminals in the adrenal medulla of the rat. The present study indicates that adrenal microdialysis is a useful method for studying the control mechanism of adrenomedullary function in the rat in vivo.