We addressed in this study, with immunocytochemical methods, the following questions: are immunoreactive enkephalins in the rat neurohypophysis stored in nerves distinct from neurosecretory nerves; where is [Met]enkephalin immunoreaction localized; does immunoreactive [Leu]enkephalin coexist with pro-enkephalin or with pro-dynorphin fragments; and are the interpretations of localization studies influenced by the choice of pre-embedding or post-embedding immunocytochemical techniques? We compared immunoreactions due to antibodies which had been used by others in previous studies, examined both lyophilized and conventionally fixed specimens, and applied pre- and post-embedding protocols. Both pre- and post-embedding stainings confirmed co-storage of immunoreactive dynorphin(1-8)-like materials with vasopressin. Immunoreactive [Met]enkephalin-like material always coexisted with oxytocin. Most of the immunoreactive [Leu]enkephalin-like material appeared to occur in oxytocin nerves; only in larger vasopressin varicosities was there some dot-like [Leu]enkephalin immunoreaction. This indicates that neural lobe [Leu]enkephalin predominantly is cleaved from a precursor which also contains [Met]enkephalin. When pre-embedding methods were modified in order to block diffusion and to enhance penetration of antibodies, enkephalin immunoreactivity was always found in typical neurosecretory varicosities with large granules. Structures previously interpreted as enkephalinergic nerve terminals contacting pituicytes most likely are neurosecretory varicosities.