The subcellular localization and the secretion of neuropeptide Y were studied in sympathetic nerve endings of bovine vas deferens. Immunostaining revealed a parallel distribution for neuropeptide Y and dopamine-beta-hydroxylase immunoreactivity in the network of varicose nerve fibers in the smooth muscle layers of the vas deferens. Following differential centrifugation and sucrose density gradient centrifugation, neuropeptide Y was found to coincide with noradrenaline in the more dense region of the gradient, where the large dense cored vesicles are found. Superfusion experiments demonstrated the release of neuropeptide Y and noradrenaline upon electrical stimulation. Furthermore, the neuropeptide Y secretion was shown to be Ca2+-dependent. We conclude that in the bovine vas deferens neuropeptide Y is only present in large dense cored vesicles of adrenergic neurons and that the peptide and noradrenaline are co-released from these vesicles in a calcium dependent manner.