Guinea-pig tracheas were perfused with recirculating modified Krebs-Henseleit solution while monitoring changes in inflow-outflow pressure difference, which is an index of trachealis muscle tone. The reactivities of the trachealis muscle to methacholine, histamine and isoproterenol applied separately to the mucosal (intraluminal, IL) or serosal (extraluminal, EL) compartments were compared, and evidence for the agonist-induced release of epithelium-derived relaxing factor (EpDRF) was sought. All agents were more potent when added to the EL compartment, but the IL/EL EC50 ratios were different: 100 for methacholine, 41 for histamine and 25 for isoproterenol. Methacholine or histamine added to the IL compartment, after the preparations were pre-contracted with the same concentration of the agonist or 30 mM KCl added EL, did not result in relaxation. Likewise, IL isoproterenol did not evoke contraction. IL KCl evoked relaxation. The results indicate that the epithelium reduces access of bronchoactive agents to the muscle, while an immediate relaxant effect of EpDRF released by agonists could not be demonstrated.