The release of a previously unrecognized smooth muscle relaxant factor from rat urinary bladder has been demonstrated by a coaxial bioassay system (1-3). In the present study using the same bioassay consisting of rat urinary bladder as the donor organ and rat anococcygeus muscle as the assay tissue, we have investigated the muscarinic receptor subtype(s) mediating the release of bladder-derived relaxant factor. The anococcygeus muscle that was placed in the lumen of the bladder was mounted under a resting tension of 1 g in an organ bath filled with Krebs-Henseleit solution, and was precontracted with phenylephrine (1-3 µM) before each experimental procedure. In this coaxial bioassay system, acetylcholine induced concentration-dependent relaxation response that was also repeated with lower potency by muscarinic M1 receptor agonist McN-A-343. The pD2 values of acetylcholine and McN-A-343 calculated from their concentration response curves were 5.91 ± 0.18 and 3.45 ± 0.34, respectively. The relaxation response to acetylcholine was inhibited by non-selective and subtype selective muscarinic receptor antagonists. The rank order of potency (pKB) was estimated as: M3 antagonist darifenacin (9.36 ± 0.11) > M3, M1 antagonist 4-DAMP (9.30 ± 0.11) > non-selective antagonist atropine (9.09 ± 0.15) > M1 antagonist telenzepine (8.56 ± 0.21) > M4 antagonist tropicamide (6.63 ± 0.17) > M2 antagonist AF-DX 116 (6.01 ± 0.21). The pKB values of subtype selective muscarinic antagonists have revealed that M1 and M3 muscarinic receptors are involved in the release of urinary bladder-derived relaxant factor.