The involvement of the M2 muscarinic receptor in contractile responses of the guinea pig trachea, guinea pig esophagus, and rat fundus was investigated. In the standard assay, oxotremorine-M elicited contractions of the trachea with an EC50 value of approximately 73 nanoM.--2- -(Diethylamino)methyl- -1-piperidinyl-acetyl--5,11- dihydro-6H-pyrido-2,3-b--1,4- benzodiazepine-6-one (AF-DX 116) at 1 and 10 microM antagonized these contractions by 2.1- and 9.0-fold increases in the EC50 value for oxotremorine-M. These effects are consistent with antagonism of an M3-mediated contractile response. In subsequent experiments, the M3 receptors were first inactivated selectively by incubation with N-(2-chloroethyl)-4- piperidinyl diphenylacetate (4-DAMP mustard) (40 nanoM) for 1 hr in the presence of AF-DX 116 (1 microM) followed by extensive washing. In 4-DAMP mustard treated trachea, oxotremorine-M elicited contractions with an EC50 value of 0.31 microM in the presence of histamine (10 microM) and forskolin (4 microM). Under these conditions, AF-DX 116 at 1 and 10 microM antagonized contractions to oxotremorine-M by 8- and 59-fold increases in the EC50, respectively, while para- fluorohexahydrosiladiphenidol(p-F-HHSiD) (0.1 microM) had no effect. These effects are consistent with a contraction being mediated by an M2 receptor. In the guinea pig esophagus and rat fundus, AF-DX 116 and p-F-HHSiD blocked contractions measured under similar conditions with magnitudes intermediate between what would be expected from an M2 and an M3 receptor, suggesting that perhaps both subtypes contribute to the overall contractile response under these conditions. In addition, contractions of the guinea pig trachea measured in the presence of histamine and forskolin were pertussis toxin sensitive. These results that, in the trachea, M2 receptors can dominate the contractile response after a majority of the M3 receptors have been inactivated, whereas in the guinea pig esophagus and rat fundus, M2 receptors may contribute to, but do not play a dominant role in the overall response.