After only 1 day of treatment with a thiamine-deficient diet and pyrithiamine, a centrally and peripherally acting thiamine antagonist, 37.5% of rats performed poorly on a standardized string test. By day 12, 89.6% of the pyrithiamine-treated rats, but only 8.1% of the pair-fed controls, had decreased string test scores. Scores for rats treated with a thiamine-deficient diet and oxythiamine, a peripherally acting thiamine antagonist, did not decrease even on the day before death. The acetylcholinesterase inhibitor physostigmine improved the low string test scores in 69.2% of trials with the pyrithiamine-treated rats, whereas neostigmine, which acts peripherally, had no effect. The effect of physostigmine was inhibited by the muscarinic blocker atropine, but not by methatropine, its peripherally acting analog. Arecoline, a direct muscarinic agonist, was as effective as physostigmine or thiamine in restoring string test performance. Nicotine had no effect and the nicotinic blocker mecamylamine did not alter the effect of physostigmine. Incorporation of [2H4]choline and [U-14C]glucose into acetylcholine was decreased by 32 and 48%, respectively, relative to pair-fed controls after 12 days of pyrithiamine treatment. Acetylcholine levels were unchanged. Thiamine deficiency induces an early functionally significant central muscarinic cholinergic lesion.