Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8-Isoprostaglandin E1and 8-isoprostaglandin E2(8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2and PGE2implicated isoprostane activity at the PGE2receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1 H-[1,2,4]-oxadiazolo[4,3- a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2did not reverse KCl tone, suggesting that voltage-dependent Ca2+influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2receptor-directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+channel activation or the direct inhibition of voltage-operated Ca2+influx or myosin light chain kinase.