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. (C) 2009 Elsevier Ltd. All rights reserved.