1. In guinea-pig and canine airway smooth muscle, there is reduced beta-adrenoceptor agonist sensitivity in tissues pre-contracted with muscarinic agonists when compared to tissues pre-contracted with other spasmogens, such as histamine or leukotriene D4. This reduced sensitivity may be the result of an interaction between muscarinic receptors and beta-adrenoceptors. In this study the effects of M2 receptor antagonism and stimulation have been investigated on the relaxant potency of isoprenaline in guinea-pig isolated tracheal smooth muscle. 2. (+)-cis-Dioxolane contracted isolated tracheal strips in a concentration-dependent manner (EC50 = 11.5 +/- 0.9 nM). The rank order of antagonist apparent affinities (with pA2 values in parentheses) was atropine (9.4 +/- 0.1) > zamifenacin (8.2 +/- 0.1) > para-fluoro-hexahydro-siladiphenidol (p-F-HHSiD, 7.2 +/- 0.1) > pirenzepine (6.5 +/- 0.1) > methoctramine (5.5 +/- 0.1). Schild slopes were not significantly different from unity. This was consistent with a role of muscarinic M3 receptors in mediating contraction. 3. In tissues pre-contracted to 3 g isometric tension using (+)-cis-dioxolane (0.2 microM, approximately EC80), the relaxant potency of isoprenaline was significantly (P < 0.05) increased by 0.3 microM methoctramine (control EC50 = 32.2 +/- 4.3 nM, plus methoctramine EC50 = 19.1 +/- 4.5 nM). This concentration of methoctramine had no effect on contractile responses to (+)-cis-dioxolane (control, EC50 = 17.6 +/- 3.2 nM, plus methoctramine, EC50 = 21.0 +/- 4.4 nM). 4 When acetylcholine (non-selective), (+)-cis-dioxolane (non-selective), L-660,863 ((+/- )-3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine, M2-selective) or SDZ ENS 163 (thiopilocarpine, mixed M2 antagonist,partial M3 agonist) were used to achieve isometric tensions of 3 g, the relaxant potency of isoprenaline ranged from 3.7 +/- 0.3 nM (SDZ ENS 163) to 49.4 +/- 3.2 nM ((+)-cis-dioxolane). Reducing the concentration of these agonists (and therefore the level of developed tension to 2 g), significantly(P<0.05) increased the relaxant potency of isoprenaline. In contrast, when histamine was used to pre-contract tissues to either 2 or 3 g (EC50 = 4.2 +/- 0.6 and 3.8 +/- 1.1 nM, respectively), there was no significant effect on the relaxant potency of isoprenaline.5. There was a slight but significant (P<0.05) reduction in the relaxant potency of isoprenaline, in tissues pre-contracted to 3 g using histamine in combination with (+ )-cis-dioxolane (30 nM). This effect was reversed by M2 receptor antagonism, using methoctramine (1 MicroM).6. These data suggest that in guinea-pig isolated trachea, the relaxant potency of isoprenaline may depend not only on the level of developed tension but also, on the level of muscarinic M2 receptor stimulation/blockade of the spasmogen inducing the tension. However, the lack of selective M2 agonist and the low M2/M3 selectivity of antagonists in this tissue do not permit definitive conclusions to be made about the role of these receptors in modulating isoprenaline potency.