The ability of the M2 muscarinic receptor to inhibit the relaxant effects of forskolin and isoproterenol was investigated in bovine trachea. In most experiments, we measured contractile responses to oxotremorine-M in smooth muscle isolated from bovine trachea in which a majority of M3 receptors were inactivated by treatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate. In the presence of histamine (20 microM), the histamine H2 antagonist cimetidine (10 microM) and forskolin (4 microM), responses to oxotremorine-M were antagonized by [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (1 microM) in a manner consistent with contractions mediated predominantly by M2 receptors. When similar experiments were conducted in the presence of isoproterenol (0.1 microM) instead of forskolin, contractions were antagonized in a manner consistent with an M3 receptor-mediated response. In similar experiments, we measured the relaxant potency of isoproterenol and forskolin against histamine-induced contractions in N-(2-chloroethyl)-4-piperidinyl diphenylacetate-treated trachea. By itself, oxotremorine-M (7.5 nM) had no contractile effect; however, it caused a substantial reduction in the relaxant potency of forskolin although having little effect on that of isoproterenol. These experiments establish that M2 receptors inhibit the relaxant effects of forskolin, but not isoproterenol. In untreated tissues, the relaxant responses to isoproterenol and forskolin were 10.8- and 14.2-fold more potent, respectively, against histamine than against oxotremorine-M-induced contractions of equal magnitude. Similarly, the maximal stimulation of cAMP accumulation elicited by isoproterenol and forskolin was inhibited 58 and 62%, respectively, in the presence of oxotremorine-M (80 nM) compared to that measured in the presence of histamine (20 microM). Analysis of the data indicated that isoproterenol elicited relaxation at concentrations well beyond those that stimulated maximal levels of cAMP accumulation. Our results indicate that part of the relaxant response to isoproterenol is mediated through a non-cAMP-dependent mechanism, and that this mechanism is largely unopposed by the M2 receptor.