Intrathecal injection of neostigmine enhances antinociception from clonidine while it counteracts clonidine-induced hypotension. This study further examined the pharmacology of neostigmine-clonidine interactions in the spinal cord and focused on the roles of muscarinic receptor subtypes and local nitric oxide synthesis. Spinal neostigmine counteracted clonidine-induced decreases in blood pressure and heart rate in conscious sheep and this effect was blocked by spinal injection of the M2 muscarinic antagonist, AFDX-116, but not by the M1 muscarinic antagonist, pirenzepine. Carbamylcholine injected spinally alone increased blood pressure and heart rate and these effects and neostigmine's hemodynamic interaction with clonidine were blocked by spinal injection of the nitric oxide synthase inhibitor, N-methyl-L-arginine. The authors also investigated antinociceptive interactions by using a mechanical pressure stimulus on the forelimb of conscious sheep. Spinal clonidine produced dose-dependent antinociception, which was enhanced by neostigmine and antagonized by N-methyl-L-arginine. NADPH diaphorase staining of sheep spinal cord revealed dense localization to the superficial dorsal horn and the intermediolateral cell column. These results suggest that counteraction of spinal clonidine-induced hypotension by neostigmine is due to stimulation of spinal M2 muscarinic receptors and synthesis of nitric oxide. Nitric oxide synthesis is also necessary for clonidine-induced antinociception in sheep.