To sequence and thereby definitively characterize corticotropin-releasing factor (CRF)-like material from a representative peripheral tissue, CRF was obtained from 76 ovine testes. The novel extraction procedure involved use of an immunoaffinity column to which a high-affinity CRF monoclonal antibody was attached as well as fast protein liquid chromatography. The complete sequence was elucidated by gas-phase sequencing, carboxyamidopeptidase digestion and cyanogen bromide cleavage. Aside from microheterogeneity at position 39, all the other amino acids were identical to ovine hypothalamic CRF. Additionally, in immunohistochemical studies in the rat, CRF was localized to the Leydig cell. These findings along with related observations by ourselves and others are compatible with the hypothesis that CRF plays a significant local role, possibly by paracrine or autocrine mechanisms.