Cellular mechanism(s) regulating atriopeptin secretion and processing by the atrial myocyte are currently unknown. Osmotic stretch of isolated atrial myocytes as well as potassium chloride depolarization were potent stimuli of atriopeptin secretion. Release was potentiated by buffering either extracellular calcium with EGTA or intracellular calcium with the intracellular chelator, BAPTA AM. Atrial release of atriopeptin was inhibited after administration of ionomycin which elevates intracellular calcium. Fetal or early neonatal ventricular myocytes actively synthesize atriopeptin. Atriopeptin secretion by ventricular myocytes was also markedly potentiated by osmotic stretch as well as KCl depolarization. Only the 126 amino acid prohormone was secreted by the stretch-stimulated atrial and ventricular myocyte. These data suggest that stretch of the myocyte plasma membrane is a major stimulus for atriopeptin secretion and that atriopeptin secretion is not stimulated by raising intracellular calcium and appears to be negatively modulated by this cation. Like the atrial myocyte, the ventricular myocyte possesses the cellular mechanism(s) necessary to secrete atriopeptin by a regulated mechanism.