Prejunctional and postjunctional muscarinic receptor subtypes were characterized in canine trachealis muscle strips. In vitro contractile responses of muscle strips to acetylcholine or electric field stimulation were determined in the absence and the presence of gallamine, pirenzepine, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP). Gallamine had no effect on the contractile response to acetylcholine but enhanced the contractile response to electric field stimulation. Pirenzepine and 4-DAMP reduced the contractile response to acetylcholine and electric field stimulation. The pA2 value for pirenzepine vs. acetylcholine [7.18 +/- 0.59 (SD)] was consistent with the affinity of pirenzepine for M2 or M3-receptors; whereas the pA2 value for 4-DAMP vs. acetylcholine (8.92 +/- 0.42) and the extremely low affinity of gallamine indicated postjunctional muscarinic receptors of the M3 subtype. The enhancement of the contractile response to electric field stimulation by gallamine suggested the presence of M2-prejunctional receptors.