The nature of the prejunctional inhibitory muscarinic receptor on cholinergic nerve endings in the rat urinary bladder was investigated by measuring stimulated endogenous acetylcholine release via high pressure liquid chromatography (HPLC), in the presence of various selective muscarinic antagonists. The rank order of potencies for the antagonists used was: atropine (-log concentration = 7.8) > 4-DAMP (4-diphenylacetoxy-N-methylpiperidine) (7.6) > tripitramine (7.3) = HHD (hexahydrodifenidol) (7.3) > pFHHSiD (p-fluoro-hexahydrosiladifenidol hydrochloride) (7.0) > himbacine (6.5) > methoctramine (5.9) > or = pirenzepine (5.8) > gallamine (4.3). A comparison of the antagonist potencies obtained, with affinity constants at muscarinic M(1) to M(5) receptors, suggests that the prejunctional inhibitory muscarinic receptor is of the M(4) receptor subtype.