Enkephalinergic innervation of the rat salivary glands was investigated by immunocytochemical techniques. Based upon immunostaining for enkephalin (ENK) and tyrosine hydroxylase (TH), 4 types of neurons could be distinguished in the submandibular ganglion: cells containing both ENK and TH (9% of all ganglion cells), cells containing only ENK (17%), cells containing only TH (4%) and cells lacking both ENK and TH (70%). Almost all of the ganglion neurons were also positive for AChE and so were most of the TH-positive cells. The ENK-positive fibers outnumbered the TH-positive fibers. Although TH-positive fibers displayed concurrent ENK immunoreactivity, fibers in the blood vessel walls were only immunoreactive for TH. Excision of the superior cervical ganglion resulted in a decrease of ENK fibers and the disappearance of most of the TH fibers from the submandibular gland. Most of the remaining ENK-positive fibers were immunonegative for TH, while the remaining TH-positive fibers were also positive for ENK. The salivary gland of the postnatal 8-week-old rats had a considerable number of ENK-positive neurons and fibers in the submandibular ganglion and acini.