The present study investigated the effects of microinjections of acetylcholine (a cholinergic agonist), physostigmine (a cholinesterase inhibitor), atropine (an antagonist of muscarinic cholinergic receptors) and hexamethonium (an antagonist of nicotinic cholinergic receptors) into the parafascicular nucleus of thalamus on the acute corneal nociception in rats. Acute corneal nociception was induced by putting a drop of 5 M NaCl solution onto the corneal surface of the eye and the number of eye wipes was counted during the first 30s. Both acetylcholine and physostigmine at the same doses of 0.5, 1 and 2 μg significantly (P < 0.05) reduced the number of eye wipes. The intensity of corneal nociception was not changed when atropine and hexamethonium were used alone. Atropine (4 μg), but not hexamethonium (4 μg) significantly (P < 0.05) prevented acetylcholine (2 μg)- and physostigmine (2 μg)-induced antinociceptive effects. The results indicated that at the level of the parafascicular nucleus of thalamus, the muscarinic cholinergic receptors might be involved in the antinociceptive effects of acetylcholine and physostigmine.