Pituitary adenylate-cyclase activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus and located in many neuronal systems in both the central and peripheral nervous system. PACAP is also found in nerve fibers innervating the pineal gland, where it stimulates the secretion of the pineal hormone, melatonin, by binding to specific PACAP-receptors located on the cell membrane of the pinealocyte. In this study we have investigated the origin of PACAP-containing nerve fibers innervating the rat pineal gland by combined retrograde tracing with Fluorogold and immunohistochemistry for PACAP. A solution of 2% Fluorogold was injected iontophoretically into the superficial pineal gland of Wistar rats, and the animals were allowed to survive for 1 week. After perfusion fixation of the rats, the location of the tracer was investigated in the brain and the sphenopalatine, otic, and trigeminal ganglia. The tracer was found in all the investigated ganglia. However, colocalization with PACAP was predominantly found in the trigeminal ganglion and only occasionally in the sphenopalatine and otic ganglia. Due to the stimulatory function of PACAP on pineal melatonin secretion, the PACAP-containing neurons of this ganglion could be considered a subset of the parasympathetic nervous system. The presence of neurons with a parasympathetic function in a ganglion that has been considered a purely sensory ganglion, is a new concept in neuroanatomy.