Muscarinic M2 receptors account for more than half the muscarinic receptor population in smooth muscles of a number of species and yet it is the smaller M3 receptor population that mediates contraction of many of these tissues. The role of the majority of M2 receptors in the control of smooth muscle tone is unclear. In guinea-pig ileal smooth muscle, an indirect contractile role (re-contraction) for M2 receptors has been demonstrated in tissues subjected to M3 receptor alkylation and stimulation of adenylyl cyclase. The present studies have employed the technique of irreversible receptor alkylation in order to investigate the role of muscarinic M2 and M3 receptors in the control of guinea-pig tracheal smooth muscle tone. Experiments were performed to determine (i) whether an indirect contractile role for M2 receptors can be demonstrated in tracheal smooth muscle as described for ileum, and (ii) whether stimulation of M2 receptors can inhibit isoprenaline-induced relaxations of histamine pre-contracted trachea after selective M3 receptor alkylation. Our results suggest (i) that there is no evidence of M2 receptor-mediated re-contraction of tracheal smooth muscle after M3 receptor alkylation and stimulation of adenylyl cyclase, but (ii) that activation of M2 receptors, after M3 receptor alkylation, has a small inhibitory effect on relaxant responses to isoprenaline in guinea-pig tracheal smooth muscle. Therefore, it appears that the major role of postjunctional muscarinic M2 receptors in guinea-pig trachea remains to be determined.