Hypothalamic tissue contains TSH-like material which is biologically, immunologically, and physicochemically similar to pituitary TSH. Immunoreactive thyroid-stimulating hormone (IR-TSH) is released from hypothalamic tissue in vitro by depolarizing concentrations of potassium or veratridine by a calcium-dependent mechanism. In the present study, we investigated the subcellular localization of IR-TSH using equilibrium density centrifugation. Tissue homogenates from intact, hypophysectomized or thyroidectomized rats were centrifuged at 150 g at 4°C for 10 min and the supernatants were layered onto continuous sucrose gradients (1.00–1.27 g/ml) and centrifuged at 100,000 g (max) for 16 h. IR-TSH in pituitary supernatants from intact and thyroidectomized rats showed high equilibrium density peaks with a modal density around 1.2 g/ml. Fractionation of the supernatant from ventral or dorsal hypothalamic homogenates resulted in abimodal distribution of IR-TSH. In supernatants from both tissues, IR-TSH containing particles were found at the top of the gradient in a low equilibrium density peak between 1.0 and 1.08 g/ml. In addition, IR-TSH containing particles were found in ventral and dorsal hypothalamic supernatants with modal densities at 1.16 and 1.25, respectively. These high density IR-TSH particles were present in tissue taken from hypophysectomized rats, and were not appreciably affected by thyroidectomy. Homogenization of the tissue in a hypo-osmotic medium disrupted the high density IR-TSH particles resulting in a single low density peak at the top of the gradient. These data suggest that hypothalamic IR-TSH is stored in membrane bound particles which have densities similar to that of secretory granul