Complete hypothalamic deafferentation in normal male rats, either limited to the median eminence, arcuate nucleus, and basal parts of ventromedial nuclei, or including, in addition, the ventromedial nuclei, dorsomedial hypothalamus, and a large part of the posterior hypothalamus, caused a decrease of resting serum TSH levels and a marked lowering of the pituitary TSH concentration. Partial frontal anterior cuts caused similar changes, whereas partial posterolateral cuts, or a complete deafferentation extended rostrally to include suprachiasmatic nuclei and caudal parts of the anterior hypothalamus, were without effect. Serum thyroxine (T4) and triiodothyronine levels in animals with the effective types of deafferentations were, after an initial short-lived decrease, within normal range despite persistent low TSH levels. Destruction of the median eminence-arcuate nucleus by electrolytic lesions in either normal or deafferented animals lowered serum TSH more than deafferentations alone, and caused a lasting decrease of serum T4 levels. We have concluded that the low serum TSH levels of the deafferented animals are caused by a partial impairment of TRF release from the median eminence-arcuate nucleus complex. This impairment seems, in turn, to be caused by the severance of the connection of the basal mid-hypothalamic structures with the more rostal parts of the hypothalamus. Our results also indicate that normal circulating levels of thyroid hormones can be maintained even at reduced serum TSH levels.