The effects of oxotremorine-M (oxo-M), a muscarinic agonist, on cyclic AMP (cAMP) accumulation in slices of the rat peripheral lung were investigated. Oxo-M stimulated cAMP accumulation in a concentration-dependent manner with an EC50 value of 4.2 microM and a maximal effect of 2.4 +/- 0.39-fold over basal. In the presence of forskolin (25 microM), the maximal effect of oxo-M was increased to 14.1 +/- 4.0-fold over basal. Forskolin alone caused a 5.9 +/- 2.2-fold increase in cAMP relative to basal; therefore, the combination of both drugs was more than additive. The effects of oxo-M on cAMP accumulation were unaffected by tetrodotoxin, indicating that the action of oxo-M was not mediated by neuronal release of neurotransmitters. Oxo-M had a small inhibitory effect on cAMP in a homogenate preparation, indicating that the stimulatory response to oxo-M in slices of the lung is not due to direct stimulation of adenylyl cyclase. Characterization of the oxo-M potentiation of forskolin-stimulated cAMP accumulation using different muscarinic antagonists yielded calculated pKB values that agreed with binding affinities for the M3 subtype. Oxo-M elicited phosphoinositide hydrolysis in the lung, and the nature of the antagonism of this response was also consistent with that expected for an M3-mediated response. cAMP accumulation in the presence of oxo-M (100 microM), forskolin (12 microM), or both drugs combined was inhibited by indomethacin (1 microM). These results demonstrate that the M3 receptor stimulates cAMP accumulation and phosphoinositide hydrolysis in the rat peripheral lung, and the mechanism for cAMP stimulation may involve arachidonic acid metabolites.