Characterization of the molecular, immunological and biological properties of a thyrotropin-stimulating hormone (TSH)-like peptide in rodent brain tissue showed similarities to its pituitary counterpart. The distribution of immunoreactivity in rodent brain was determined by radioimmunoassay in both intact and hypophysectomized animals. Easily detectable but smaller quantities of immunoassayable TSH were present in formalin fixed, acetone-stored brains from Macaca mullata. No change in the level of this TSH-like peptide was observed in most rat brain parts, although the hypothalamus did show a significant drop in hormone level after removal of the pituitary. Extracts of brain containing TSH-like material restored thyroid histology to normal when administered to hypophysectomized rats. Tissue cultured neural cells from both intact and hypophysectomized rats released a TSH-like material into the medium over a 30-day time period. Sodium /-thyroxine suppressed TSH release from pituitary monolayers but did not alter TSH release from brain derived cells that were similarly cultured. Surgical thyroidectomy resulted in a striking rise in serum TSH concentrations with no alteration in the content of brain TSH.