Using a combination of horseradish peroxidase (HRP) histochemistry and immuno-beta-galactosidase staining, and pre-embedding immuno-electron microscopy, the present study was intended to re-examine the origins of enkephalin-like immunoreactivity (ENKI) in the rat lateral septum (LS), and to show the fine structures of septal ENKI fibers. Following HRP injection into the LS, double-labeled cells which contained a homogeneous blue reaction product of ENKI and a black or brown granular reaction product of retrogradely transported HRP were identified in 4 discrete brain regions: perifornical hypothalamic area at the level of the paraventricular nucleus (PeF); posterior part of the anterior hypothalamic nucleus (AHP); bed nucleus of the stria terminalis, medial division, posterolateral part (BSTMPL); and dorsal hypothalamic area (DA). Immuno-electron micrographs demonstrated that some of the ENKI terminals in the LS form synapses with the soma and dendrites of septal neurons devoid of ENKI, though ENKI dendrites postsynaptic to non-immunoreactive terminals were also seen in the LS. These findings suggest that a large proportion of septal ENKI fibers have their origins in the above regions (PeF, AHP, BSTMPL, DA) different from the previously considered one, and they further provide a morphological basis for the postsynaptic inhibitory effects of enkephalins on septal neurons.