This study was undertaken to assess the importance of muscarinic receptor subtypes in equine airway disease. Smooth muscle strips from the mid‐cervical portion of the trachea of horses were placed in tissue baths and isometric contractile force was measured. Active force was measured in response to metacholine and the selective muscarinic receptor agonists McN‐A‐343 (M1‐selective) and pilocarpine (M2‐selective) in cumulative concentrations (10‐9M through 10‐3M), with and without preincubation with three or four concentrations of the selective muscarinic receptor antagonists pirenzepine (M1‐selective), methoctramine (M2‐selective), and 4‐DAMP (M3‐selective). The tissues contracted in response to all muscarinic agonists. The maximum responses (mean ± sem) were 86.7 ± 6.2 g for metacholine, 27.1 ± 2.5 g for McN‐A‐343 and 37.6 ± 3.5 g for pilocarpine. Preincubation with the selective muscarinic receptor antagonists resulted in dose‐dependent rightward shifts of the concentration‐effect curves for metacholine. pA2values (means ± sem) were 8.88 ± 0.30 for 4‐DAMP, 6.53 ± 0.38 for methoctramine, and 6.72 ± 0.31 for pirenzepine. Preincubation with 10‐7M 4‐DAMP resulted in a rightward shift of the concentration‐effect curves for McN‐A‐343 and pilocarpine. These results indicate that the most important muscarinic receptor mediating contraction of equine tracheal smooth muscle is of the M3‐type. Therefore relatively low concentrations of a M3‐selective muscarinic receptor antagonist will inhibit acetylcholine‐induced contraction of equine airway smooth muscle.