Neurointermediate lobes (NILS) of the pituitary glands of adult male Sprague-Dawley rats were incubated in media in the presence of corticotropin-releasing factor (CRF), a stimulator of proopiomelanocortin (POMC) peptide release. Alpha-helical CRF, a peptide known to inhibit CRF induced POMC peptide release from the anterior pituitary, was incubated with NILS for a period of 90 min, to study its potential ability to modulate peptide release from the intermediate lobe. The alpha-helical peptide reduced beta-endorphin release from NILS, as measured by radioimmunoassay (RIA), when added for the entire incubation, or when added 30 min after start of the incubation period, with CRF present. Alpha-helical CRF alone reduced beta-endorphin release, as compared to control or CRF-treated lobes. Ultrastructural examination of intermediate lobes fixed at the end of incubations revealed a reduction in the numbers of Golgi-associated dense granules, an indicator of new peptide synthesis, in intermediate lobe tissue treated with alpha-helical CRF alone, both peptides together, or with CRF followed by alpha-helical peptide. The in vitro studies demonstrate the effectiveness of the antagonist peptide on intermediate lobe peptide secretion, thereby extending its effects to both POMC-secreting areas of the pituitary gland.