Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-3H]scopolamine chloride ([3H]NMS) binding to all three tissues was saturable and of high affinity, with KD values ranging between 1.6+/-0.7 and 1.9+/-0.3 nmol/L. [3H]NMS binding identified a higher density of total muscarinic receptors (fmol/mg protein) in the trachea (720+/-59 nmol/L) than in bronchi (438+/-48 nmol/L) or lung (22 +/- 3 nmol/L). Competitive binding studies using [3H]NMS and the unlabelled subtype-selective antagonists pirenzepine and telenzepine (M1), methoctramine and himbacine (M2), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3), tropicamide (M4) and mamba toxin (MT-3) (M4) indicated the presence of at least three muscarinic receptor subtypes in peripheral lung tissue (50:40:24-28%: M2>M3>M1), whereas in bronchus and trachea M2 subtypes (87-90%) predominated over M3 (14-22%), and M1 subtypes were lacking. No differences were found between tissues in high-affinity binding sites for carbachol in the absence (31-36%) or presence of guanosine 5'-triphosphate (GTP) (approximately 100%). Western blotting for G-protein alpha-subunits showed a much more robust expression of G(alphai1/2) in the trachea (with highest receptor density) than in the lung or bronchi, whereas G(alphas)-protein was dominantly expressed in bronchus. Concomitantly, carbachol inhibited isoproterenol- and GTP-stimulated adenylyl cyclase activity with increasing muscarinic receptor expression (trachea > bronchi > lung). We conclude that the expression and signalling pathways of muscarinic receptors in the equine respiratory tract are segment-dependent. These receptors might contribute to the pathogenesis of COPD in the horse and could provide potential drug targets for the therapeutic use of anticholinergics in this species.