We have previously reported the ability of isoprostanes to induce airway hyperresponsiveness (AHR). In this study, we examined the signaling mechanisms underlying that phenomenon using the standard muscle bath technique. Responses to a threshold concentration of Cch (3x10^-9 M) were significantly augmented by pretreatment for 20 minutes with 15-E2t-IsoP (10^-6 M): this AHR was obliterated in tissues pretreated with the selective-ROCK inhibitor Y27632 added 20 minutes prior to the isoprostane, but not by cyclopiazonic acid (CPA). Increasing the concentration of Cch to 3x10^-8 M (still considerably less than the half-maximally effective concentration of carbachol) evoked larger contractions which were also augmented significantly by 15-E2t-IsoP (Fig. 2A, right): this AHR was completely abolished in tissues pretreated with CPA as well as those pretreated with Y27632. We noted, however, that Y27632 and CPA profoundly effect baseline tone and the cholinergic response per se, which confounds the interpretation of the data summarized above. We therefore modified the protocol by using combinations of CCh and blocker (CPA, Y27632 or nifedipine) which were equieffective. In this way, we found AHR could not be demonstrated under conditions in which Rho/ROCK signaling or Ca²⁺-release were abolished (by Y27632 and CPA, respectively). Likewise, other autacoids which act through G-protein coupled receptors via Rho/ROCK and Ca²⁺-release (serotonin; histamine) mimicked this effect of isoprostane, whereas bradykinin did not. We conclude that isoprostane-induced AHR is mediated in part through an action on Rho/ROCK signaling. This novel finding may contribute to a better understanding of the mechanisms underlying airway hyperresponsiveness and asthma.