The inhibitory effect of the selective M 3 musarinic acetylcholine receptor antagonist, 4diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP, 0.1-10 µM) on nicotine (100 µM)-induced nitrergic relaxation was investigated in comparison to d-tubocurarine (0.1-10 µM) and hexamethonium (0.1-10 µM) by using phenylephrine (1 µM)-precontracted rat anococcygeus muscles in vitro. Nicotine produced a 60.1 ± 2.4% (n = 40) inhibition of phenylephrine precontractions. But this relaxant response was at a significantly lower magnitude of 20.2 ± 4.6% (n = 18, P < 0.01 vs. control) in the presence of the nitric oxide synthase (NOS) blocker N G -nitro-L-arginine methyl ester (L-NAME, 30 µM), and it was 26.5 ± 5.5% (n = 8, P < 0.01 vs. control) in the presence of the soluble guanylate cyclase inhibitor methylene blue (30 µM). However, aminoguanidine (100 µM), a relatively selective blocker of the inducible nitric oxide synthase (iNOS), had no significant effect. Similarly, other iNOS inhibitors such as dexamethasone (5 mg/ kg) or L-canavanine (100 mg/kg) did not modify contractile nor relaxant responses when they were given in vivo, concomitantly with Escherichia coli endotoxin (1 mg/kg, ip) 4 h before the isolation of the tissues. 4-DAMP, hexamethonium, and d-tubocurarine inhibited nicotine-induced relaxation in a concentration-dependent manner with the following order of potency: 4-DAMP > hexamethonium > d-tubocurarine with IC 50 values being 0.47 ± 0.04 µM, 0.75 ± 0.06 µM, and 1.02 ± 0.05 µM, respectively. Therefore, it was concluded that the selective M 3 muscarinic acetylcholine receptor antagonist 4-DAMP also possesses potent antagonistic action on nicotinic receptors of peripheral nitrergic neurons that innervate the rat anococcygeus muscle.