The influence of the site of action of thyrotropin-releasing hormone (TRH) on the production of hyperglycemia was studied in rats by comparing the effectiveness of TRH administered by different routes. Administration of TRH (5 micrograms) into the lateral hypothalamus (LH) produced a hyperglycemia with a peak elevation of blood glucose of 140 mg/dl. Injection of 5 micrograms TRH into the ventromedial hypothalamus (VMH) produced a blood glucose elevation of 44 mg/dl, while injection of the same dose of TRH into the anterior hypothalamus (AH) produced a blood glucose elevation of 23 mg/dl. These findings indicate an LH site of action of TRH. Indeed, intra-LH administration of TRH (1-10 micrograms) caused dose-related increases in blood glucose. Administration of acetylcholine into the same site was also shown to induce hyperglycemia. The hyperglycemic effects of TRH and acetylcholine were antagonized by previous treatment of the LH site with atropine, a cholinergic receptor antagonist. Furthermore, the TRH-induced hyperglycemia was not observed or was greatly reduced in spinal rats or in adrenalectomized rats. The results indicate that TRH may act through the cholinergic receptor mechanisms within the LH region to induce hyperglycemia by promoting an increase in the sympathetic-adrenal medullary efferent activity.