In addition to their direct contractile effects, histamine (Hist), serotonin [5-hydroxytryptamine (5-HT)], and leukotriene (LT) D₄, in low concentrations, dramatically augment electrical field stimulation (EFS)-induced smooth muscle contractions in equine airways. To determine the mechanism of their action, we studied, in trachealis strips, the effect of these mediators on both cholinergically induced tension and the release of ACh from cholinergic nerves. All three mediators synergistically augmented the contraction of the trachealis that was due to release of endogenous ACh, i.e., EFS-induced contraction. These same mediators caused only a small but parallel shift of the ACh concentration-response curve. Comparison of the mediator effects on the responses to endogenous and exogenous ACh suggested a prejunctional effect. However, release of ACh was augmented only by Hist and 5-HT but not by LTD₄. Hist-induced contraction of trachealis was abolished by pyrilamine (H₁-receptor antagonist) but not by ranitidine (H₂-receptor antagonist), whereas thioperamide (H₃-receptor antagonist) shifted the Hist response curve to the left. The augmenting effect of Hist on EFS-induced contraction was abolished by pyrilamine and unaffected by ranitidine or thioperamide. We conclude that inflammatory mediators can increase endogenous cholinergic responses of equine airways via both prejunctional and postjunctional mechanisms. LTD₄ acts solely on smooth muscle, whereas 5-HT and Hist additionally act on neuronal receptors to facilitate release of ACh. Excitatory effects of Hist, i.e., direct contractile effect, and augmentation of endogenous cholinergic response are both mediated via H₁ receptors, whereas the inhibitory H₃ receptors partially oppose the direct contractile effect of this mediator.