Muscarinic receptors play a major role in gallbladder function, although the muscarinic receptor(s) mediating smooth muscle contractility is unclear. This study compared smooth muscle contractile responses to carbamylcholine (10(-7)-10(-3) M) in isolated gallbladder from wild-type and M(2), M(3), and M(4) receptor knockout mice. Carbamylcholine-induced contraction in gallbladder was associated with tachyphylaxis and the release of a cyclooxygenase product because indomethacin (10(-6) M) inhibited carbamylcholine-induced contraction. The M(3) receptor was the major muscarinic receptor involved in contraction because carbamylcholine-induced contractility was inhibited in gallbladder from M(3) receptor knockout mice. Furthermore, the muscarinic receptor antagonists 11-[[[2-diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) and pirenzepine dextrally shifted contraction to carbamylcholine in gallbladder from wild-type, M(2), and M(4) receptor knockout mice, with affinities consistent with M(3) receptor interaction. In addition, maximal contraction to carbamylcholine was reduced in gallbladder from M(2) receptor knockout mice and affinities for AF-DX 116 and pirenzepine in gallbladder from M(3) receptor knockout mice were consistent with their affinities at M(2) receptors. In M(4) receptor knockout mice, contraction to carbamylcholine was dextrally shifted, although the affinities for AF-DX 116 and pirenzepine in gallbladder from M(2) or M(3) knockout mice were not similar to their affinities at M(4) receptors. The M(4) receptor may serve as an accessory protein necessary for optimal potency of M(2) and M(3) receptor-mediated responses. Thus, muscarinic receptor knockout mice provided direct and unambiguous evidence that M(3), and to a lesser extent, M(2) receptors are the predominant muscarinic receptors mediating gallbladder contractility, and M(4) receptors appear necessary for optimal potency of carbamylcholine in gallbladder contraction.