This investigation was carried out on the distribution of enkephalin-containing nerve fibres and terminals in the region of the nucleus basalis magnocellularis (NBM) of the rat. At the light microscope (LM) level, enkephalin-immunoreactive sites and endogenous choline acetyltransferase (ChAT) were demonstrated by employing the two-colour immunoperoxidase staining technique, using highly specific monoclonal antibodies against enkephalin and ChAT. A pharmacohistochemical procedure to reveal acetylcholinesterase (AChE)-synthesizing neurons combined with the peroxidase-antiperoxidase (PAP) immunocytochemical technique to detect endogenous enkephalins, provided ultrastructural data on the relationships of neuronal elements containing AChE and enkephalins in the region of the NBM. At the LM level, cholinergic neurons of the NBM were surrounded by a dense network of enkephalin-immunoreactive nerve fibres. Electron microscopic (EM) observations of histochemically characterized structures, that were first identified in the LM, revealed that intensely AChE-stained structures in the region of the NBM received sparse synaptic inputs from enkephalin immunoreactive terminals. Synaptic inputs of immunoreactive terminals onto intensely AChE-stained neuron cell bodies were not detected. Synaptic contacts onto proximal AChE-positive dendrites were sparse, but the density increased on more distal regions of the dendrites. All immunoreactive boutons studied established symmetrical synaptic contacts with AChE-positive post-synaptic structures. The pattern of the synaptic input to these cells differs strikingly from that onto typical globus pallidus neurons. The perikarya and dendrites of the latter neurons were characteristically ensheathed in immunoreactive synaptic boutons. Results are consistent with the view that enkephalin-like substances in the rat might be synaptic transmitters or neuromodulators in the area of the NBM and that cholinergic neurons of the NBM (Ch4) are integrated into the circuitry of the basal ganglia. Enkephalins may play an important role regulating the extrinsic cholinergic innervation of the neocortex.