Spinal neostigmine produces analgesia in chronically prepared rats, but not in sheep. However, since pain itself activates bulbospinal inhibitory pathways, neostigmine may be more effective in the postoperative period. We examined in sheep the antinociceptive effect of intrathecal neostigmine in the acute postoperative period and determined the muscarinic receptor subtype activated by neostigmine. A cervical intrathecal catheter was inserted via a laminotomy in 14 sheep that then received, in random order 1 mg of spinal neostigmine or saline on postoperative Day 1 and the other injection on postoperative Day 2. Three additional sheep received, on separate days, intrathecal neostigmine alone or with the muscarinic receptor subtype-specific antagonists pirenzepine (M1) 2 mg or AFDX-116 (M2) 2 mg. Antinociception was tested using a mechanical stimulus after each injection. Baseline withdrawal threshold did not change postoperatively. Intrathecal neostigmine, but not saline caused antinociception on both of the first two postoperative days. In contrast, intrathecal neostigmine caused no antinociception in another similar study performed at least 5 days after surgery. Pirenzepine, but not AFDX-116, abolished antinociception from neostigmine, suggesting an action on M1 subtype muscarinic receptors. Intrathecal neostigmine is antinociceptive in sheep during the acute postoperative period, and these data suggest that spinal cholinergic tone, and hence intrathecal neostigmine's analgesic effect, may be enhanced during the acute postoperative period.