The purpose of this study was to test the hypothesis that H2O2 decreases the amount of force produced by a given intracellular Ca2+ concentration (i.e., the Ca2+ sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H2O2 and then permeabilized with 10% Triton X-100 to assess the persistent effects of H2O2 on Ca2+ sensitivity. Experiments in which H2O2 was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca2+ sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H2O2 was added to permeabilized strips. H2O2treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca2+ sensitivity may occur.