A substance exhibiting potent activity in stimulating the release of prolactin from bullfrog (Rana catesbeiana) pituitary in vitro was isolated from an acid extract of bullfrog hypothalami by gel-filtration chromatography (Sephadex G-15), ion-exchange chromatography (Mono-S HR 5/5), and reverse-phase high-performance liquid chromatography (TSK-gel ODS-120T). Its amino acid composition was similar to that of synthetic thyrotropin-releasing hormone (TRH). Radioimmunoassay confirmed that the substance had TRH immunoreactivity. Moreover, it exhibited the same chromatographic behavior as that of synthetic TRH. These results clearly indicate that the isolated hypothalamic substance is TRH, and that it is the major prolactin-releasing factor present in the bullfrog hypothalamus.