The effect of H2O2 on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actin-activated rate of Pi release were measured by single-turnover kinetics. H2O2 treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, ∼50% of the molecules lost the ability to bind to ATP. H2O2 treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H2O2-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H2O2-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mM ATP, H 2O2 caused a ∼50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H2O 2 directly affects the force generating complex. Dithiothreitol treatment reversed the H2O2 inhibition of the maximal force by ∼50%. These data, when compared with the in vitro kinetic data, are consistent with a H2O2-induced loss of functional myosin heads in the muscle. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.