Neuropeptide Y co-exists with noradrenaline in the majority of the sympathetic nerves supplying cerebral blood vessels. However, after sympathectomy in the rat the number of cerebrovascular neuropeptide Y nerve fibers are only reduced in number despite a complete disappearance of the adrenergic markers. The origin of these non-sympathetic neuropeptide Y fibers was studied by nerve transections and retrograde axonal tracing utilizing True Blue. Three days after bilateral superior cervical sympathectomy, the number of neuropeptide Y-containing nerve fibers decreased to about 40% of that in non-treated animals. One week after True Blue application on the proximal portion of the middle cerebral artery, the tracer accumulated in neurons of the sphenopalatine, otic, and internal carotid ganglia. Of these cells 80%, 95% and 5%, respectively, were neuropeptide Y-positive. Some of the True Blue/neuropeptide Y-positive cells displayed immunoreactivity for vasoactive intestinal polypeptide and some were positive for choline acetyltransferase. Two weeks after bilateral removal of the sphenopalatine ganglion or transection of postganglionic fibers from the ganglion reaching the pial vessels through the ethmoidal foramen, together with subsequent sympathectomy, no neuropeptide Y-containing nerve fibers could be observed on the anterior cerebral and internal ethmoidal artery or the distal portion of the middle cerebral artery, whereas a few nerve fibers remained on the proximal portion of the middle cerebral artery, internal carotid artery, and the rostral portion of the basilar artery. In conclusion, neuropeptide Y in cerebrovascular nerves is co-stored not only with noradrenaline in sympathetic nerves from the superior cervical ganglion, but also with acetylcholine (reflected in the presence of choline acetyltransferase) and vasoactive intestinal polypeptide in parasympathetic nerves originating in the sphenopalatine, otic, and internal carotid ganglia.