We have investigated the muscarinic receptor subtype(s) mediating the release of urinary bladder-derived relaxant factor that is demonstrated by a coaxial bioassay system. Acetylcholine-induced relaxation of a precontracted anococcygeus muscle mounted within the bladder was considered as an evidence for the release of this factor. M(1)-muscarinic agonist McN-A-343 and the cholinesterase inhibitor physostigmine also elicited relaxation responses in the coaxial bioassay besides acetylcholine. Acetylcholine-induced relaxation was antagonized by the subtype-selective muscarinic antagonists (pK(B)): M(3)-antagonist darifenacin (9.36+/-0.11), M(3)/M(1)-antagonist 4-DAMP (9.30+/-0.11), M(1)-antagonist telenzepine (8.56+/-0.21), M(4)-antagonist tropicamide (6.63+/-0.17) and M(2)-antagonist AF-DX 116 (6.01+/-0.21). The pK(B) values of these antagonists have suggested that stimulation of M(1)- and M(3)-muscarinic receptors in the bladder wall mediates the release of urinary bladder-derived relaxant factor. In addition, McN-A-343, by activating the facilitatory M(1) receptors and physostigmine by inhibiting the acetylcholinesterase may induce the release of this factor through endogenous acetylcholine in the coaxial bioassay system.