1. The purpose of the present experiments was to study the underlying mechanisms responsible for the relaxant action of endothelin-1 (ET-1) in the guinea-pig trachea in vitro. 2. In tracheal strips precontracted (60-70% of the maximum) with carbachol, ET-1 (1-100 nM) evoked slowly developing concentration-dependent relaxations. Preincubation of the tissues with the thromboxane A2/prostaglandin H2 receptor antagonist, BM 13505 (5 microM) significantly potentiated the relaxant response to ET-1. 3. Removal of the epithelium changed the response of precontracted tracheal preparations to ET-1 from a relaxation to a sustained contraction. 4. ET-1-induced relaxations were abolished by methylene blue (10 microM) and were almost completely attenuated by oxyhaemoglobin (5 microM) and NG-monomethyl-L-arginine (L-NMMA, 100 microM), an inhibitor of nitric oxide synthesis, but were not altered by indomethacin (10 microM). 5. In tracheal strips under passive tension, ET-1 (1-100 nM) elicited dose-dependent contractions. The sensitivity of tissues to ET-1 was significantly enhanced by removal of the epithelium (apparent EC50 values were 28.1 +/- 4.1 and 12.5 +/- 0.8 nM in intact and rubbed trachea, respectively, n = 7, P < 0.01). 6. Preincubation of intact tracheal strips with methylene blue, oxyhaemoglobin or L-NMMA did not mimic the effect of epithelium removal on ET-1-induced contractions. 7. There was a concentration-dependent increase in thromboxane A2 but not in PGE2 and prostacyclin release from intact tracheal strips following stimulation with ET-1 (5-100 nM). 8. These results show that ET-1 exerts a dual action on guinea-pig isolated trachea: it evokes contractions at low resting tone, whereas it induces relaxations at higher resting tone. The relaxant action of ET-1 may be mediated by nitric oxide released from epithelial cells and resultant activation of smooth muscle guanylate cyclase.