Immunoreactive thyroid-stimulating hormone (IR-TSH) has been detected in the hypothalamus and is released in vitro by a calcium-dependent mechanism when the tissue is depolarized. Recently, immunocytochemical studies have revealed that IR-TSH is present in thyrotropes in the pars tuberalis. Therefore, because these thyrotropes are associated with the median eminence, the area with the highest concentration of IR-TSH, it is of interest to determine if ‘hypothalamic’ IR-TSH is from neural or pituitary cells. We addressed this issue by studying the effects of hypophysectomy, thyroidectomy, or chronic administration of triiodothyronine (T3) or thyroxine (T4) on the distribution and in vitro release of IR-TSH in the hypothalamus. We reasoned that, if hypothalamic IR-TSH is dependent on the thyrotropes of the pars tuberalis, then changes in hypothalamic IR-TSH concentration and release should be parallel to those measured in pituitary extracts. IR-TSH was measured in tissue extracted in ice-cold 2% NaCl, with a final pH of 4.5. For the in vitro studies, tissues were incubated for 20-min periods in Krebs-Ringer bicarbonate buffer at 37 °C. In untreated rats, the concentration of IR-TSH is greater in the ventral than the dorsal portion of the hypothalamus (39.3 ± 8.2 vs. 4.0 ± 1.5 ng/mg wet wt). Upon finer dissection of the hypothalamus into median eminence and anterior, middle, and posterior portions of the remainder, IR-TSH was only detectable in the middle hypothalamus (5.3 ± 1.5 ng/mg), and the median eminence (149 ± 41 ng/mg). After thyroidectomy, pituitary IR-TSH content and the spontaneous in vitro release decreased, while the hypothalamic IR-TSH concentration and in vitro release were unaffected. Similarly, the hypothalamic IR-TSH concentration and release were not altered after hypothysectomy. Chronic treatment with T4 (5 µg/100 g/day) or T3 (1.25 µg/100 g/day) decreased pituitary IR-TSH content, while hypothalamic content was increased or unaffected, respectively. Spontaneous and stimulated IR-TSH release from the pituitary decreased after chronic treatment with T3 or T4, whereas the release of IR-TSH from hypothalamic tissue was unaffected. In addition, in vitro incubation of hypothalami with thyrotropin-releasing hormone (10–5M) failed to stimulate the release of IR-TSH from hypothalamic tissue. These data indicate that hypothalamic IR-TSH does not simply represent the concentration of IR-TSH in thyrotropes of the pars tuberalis, and that the factors which regulate the synthesis and release of hypothalamic IR-TSH are different from those which regulate the release of IR-TSH from t