We examined the ionic mechanisms underlying the responses of canine trachealis to superoxide (generated in vitro by using xanthine oxidase or added exogenously) and peroxide (generated spontaneously in vitro by the dismutation of superoxide or added exogenously). Although neither had any effect on resting tone, both triggered relaxations in carbachol-precontracted tissues. These relaxations were eliminated by catalase but were much less sensitive to the hydroxyl radical scavenger dimethylthiourea, indicating they were mediated primarily by peroxide. These relaxations were decreased in magnitude and/or slowed by nifedipine (10− 6 M), ouabain (10− 6 M), or tetraethylammonium (25 mM), but not by 4-aminopyridine (5 mM), and were small or absent in tissues precontracted with 30 mM KCl. Finally, peroxide triggered membrane hyperpolarization and elevated cytosolic concentration of Ca2+ (primarily via release from the internal store). Thus peroxide-mediated relaxations seem to involve Ca2+ release, opening of Ca2+-dependent K+ channels, hyperpolarization, closure of Ca2+ channels, and relaxation. In addition, some other free radical (hydroxyl radical?) may activate the Na+-K+ pump, also hyperpolarizing the membrane and causing relaxation.