A sheep antiserum to somatostatin was used to develop RIA and immunoaffinity chromatography methods for the study of immunoreactive somatostatin (IRS) in brain tissue. IRS extracted from rat median eminence, anterior hypothalamic-preoptic area, amygdala, and parietal cortex bound reversibly to immunoaffinity columns, providing a technique for concentration and partial purification. Immunoaffinity purified IRS from each of the four brain regions eluted as four peaks on gel filtration chromatography. Each peak possessed biological activity, as determined by inhibitory effects on the release of GH from cultured rat anterior pituitary cells. No differences were detected by the methods employed between IRS from the anterior hypothalamic-preoptic area, which is rich in IRS-containing neuronal cell bodies, and that from the median eminence, where IRS is localized predominantly in nerve terminals.