The cold-stimulated thyrotropin (TSH) secretion in male rats was suppressed by muscarinic agonists, i.e. Oxa-22, McN-A-343 (an M1 agonist), oxotremorine (an M2 agonist) and methacholine (a quaternary compound). The inhibitory effect of Oxa-22 was antagonized by atropine, butylscopolamine and glycopyrrolate as well as by pirenzepine, an M1 antagonist and AF-DX 116, a new M2 antagonist. Various muscarinic antagonists were not active when given alone. Cytisine, a peripheral nicotinic agonist, was not active but nicotine significantly suppressed the cold-stimulated TSH secretion. Its effect was counteracted by mecamylamine but not by hexamethonium. The thyrotropin releasing hormone (TRH)-induced TSH secretion was not inhibited by Oxa-22, nicotine or methacholine. These results show that irrespective of the receptor subtype (muscarinic1 or muscarinic2, nicotinic), cholinergic activation inhibits the cold-stimulated TSH secretion. The results also suggest that this inhibitory effect is at the hypothalamic rather than the anterior pituitary level. The muscarinic action seems to occur outside the blood-brain barrier but the nicotinic action occurs inside this barrier.