Previous reports of somatostatin's atropine-sensitive negative inotropic effect on cardiac function prompted the present studies to characterize the molecular forms and actions of somatostatin in the canine heart. Radioimmunoassay of cardiac extracts revealed concentrations of somatostatin-like immunoreactivity ranging from 0.50 +/- 0.13 pmol/g tissue (means +/- SE, n = 6) in the pulmonary artery to 0.78 +/- 0.23 pmol/g tissue in the right ventricle. On gel filtration of the extracts, two major molecular forms of somatostatin-like immunoreactivity were elicited, the predominant one coeluting with somatostatin-14 and a minor peak corresponding to somatostatin-28. Experiments performed with slices of atrial septum indicated that somatostatin-14 (10(-10) to 10(-7) M) stimulated the release of acetylcholine in a dose-dependent manner. This action of somatostatin-14 was additive with K+-evoked acetylcholine release, unaffected by hexamethonium, and blocked by tetrodotoxin, suggesting mediation via a nonnicotinic postganglionic neural pathway. Our studies lead us to conclude that somatostatin may function as a neurotransmitter in the heart, which exerts its negative inotropic action by promoting the release of acetylcholine.