1. The muscarinic receptor subtype that mediates contraction of guinea-pig trachea, in the presence and absence of epithelium, to acetic and carbamic acid choline esters was determined by use of preferential muscarinic receptor antagonists: pirenzepine (M1 receptor), methoctramine (M2 receptor) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3 receptor). 2. Acetylcholine (ACh), methacholine (MeCh), carbachol (CCh), bethanechol (BeCh) and oxotremorine induced concentration-dependent contraction of guinea-pig isolated tracheal strips in the presence and absence of epithelium. Contraction to acetic choline esters (ACh and MeCh) was augmented by removal of the epithelium, whereas contraction to carbamic acid choline esters (CCh and BeCh) and oxotremorine was not influenced by removal of the epithelium. 3. Pirenzepine, methoctramine and 4-DAMP caused parallel rightward displacements of the concentration-contraction curves to the muscarinic agonists. The pA2 values (determined from Arunlakshana-Schild graphs) for pirenzepine and 4-DAMP in guinea-pig trachea in the presence of epithelium were: ACh as the agonist, 7.6 and 9.0, respectively; CCh as the agonist, 7.6 and 9.1, respectively. The apparent pKB values for methoctramine with the same system were: ACh as the agonist, 5.6; CCh as the agonist, 5.6. Similar values were obtained with MeCh, BeCh and oxotremorine as the agonists. These values were agonist- and epithelium-independent. 4. It is concluded from the pA2 and apparent pKB values obtained for the muscarinic receptor antagonists used in this study that contraction of guinea-pig isolated trachea, with and without epithelium, to both acetic and carbamic acid choline esters is mediated via the muscarinic M3 receptor subtype.Differential contractile responses of guinea-pig trachea to acetic and carbamic acid choline esters upon the mechanical removal of the epithelium may not be explained by activation of different muscarinic receptor subtypes by these agonists.