The present study was design to determine the role of prostaglandin E2 and I2 in the responses of isolated canine airways to H2O2. Rings of canine third-order bronchi, some of which had undergone mechanical denudation of the epithelium, were suspended in organ chambers; isometric tension was recorded. During contractions to acetylcholine, H2O2 induced concentration-dependent relaxations. The relaxations were attenuated significantly by indomethacin, acetylsalicylic acid, and methylene blue. H2O2 increased the release of prostaglandin E2 and 6-keto-prostaglandin F1 alpha and the content of adenosine 3',5'-cyclic monophosphate (cAMP). These effects were abolished by indomethacin or methylene blue. H2O2 did not affect the content of guanosine 3',5'-cyclic monophosphate significantly. These observations suggest that 1) H2O2 relaxes canine bronchial smooth muscle and 2) elevation of tissue content of cAMP induced by prostaglandin E2 and I2 may be involved. These phenomena did not appear to be modulated by the respiratory epithelium, since H2O2-induced relaxations and increases in the release of PGE2 and 6-ketoprostaglandin F1 alpha were similar in preparations with and without epithelium. However, after treatment with methylene blue, H2O2 induced contractions only in preparations with epithelium. These epithelium-dependent contractions were not affected by inhibitors of cyclooxygenase and lipoxygenase.