Purified adult rat Leydig cells were found to produce gastrin-releasing peptide (GRP) by radioimmunoassay (RIA). Gel chromatography of the extracted material showed a single peak of GRP immunoreactivity. Further high pressure liquid chromatography (HPLC) analysis resolved the extract into two peaks that closely resembled the C-terminal fragment of GRP, GRP18-27 and GRP14-27. Immunohistochemical studies revealed specific staining for GRP in the Leydig cells of adult rat testis. These results demonstrate, by a number of independent criteria, that rat Leydig cells contain substances which behave like authentic GRP-like peptides. Since the peptides appear to be of local origin, a paracrine function within the rat testis is suggested.