Thyroidectomy had a pronounced effect on basal and TRF stimulated TSH release and on basal and TRF stimulated 614C glucose oxidation by rat pituitaries in vitro. Both basal TSH release and basal glucose oxidation were markedly reduced while TRF stimulation of both parameters was increased. Pituitary TSH levels decreased sharply for 3 days after thyroidectomy increasing thereafter to 64% of the euthyroid level at 39 days. In contrast, basal TSH release in vitro decreased with time reaching a minimum value 21 days after thyroidectomy. After an initial drop observed at 3 days, the absolute amount of TSH released increased with time but remained less than in euthyroid glands. The highest magnitude of TRF stimulation of both glucose oxidation and TSH release in vitro occurred approximately 21 days after thyroidectomy and after 90 min of incubation. The TRF effect on glucose was not observed with any of the constituent amino acids of this tripeptide nor with equimolar amounts of a combination of the three amino acids. 214C Pyruvate oxidation was also significantly increased by TRF. TSH release was maximally stimulated by concentrations of TRF causing no alteration of glucose oxidation and the effect on glucose oxidation occurred when protein synthesis was blocked by cycloheximide. The results obtained demonstrated that TSH release and glucose oxidation are related but are separable, thus probably not critically dependent upon each other. The TRF increase in glucose oxidation does not appear to be dependent upon an effect on TSH synthesis.