Oxidative modification of contractile proteins is thought to be a key factor in muscle weakness observed in many pathophysiological conditions. In particular, peroxynitrite (ONOO−), a potent short-lived oxidant, is a likely candidate responsible for this contractile dysfunction. In this study ONOO− or 3-morpholinosydnonimine (Sin-1, a ONOO− donor) was applied to rat skinned muscle fibers to characterize the effects on contractile properties. Both ONOO− and Sin-1 exposure markedly reduced maximum force in slow-twitch fibers but had much less effect in fast-twitch fibers. The rate of isometric force development was also reduced without change in the number of active cross bridges. Sin-1 exposure caused a disproportionately large decrease in Ca2+ sensitivity, evidently due to coproduction of superoxide, as it was prevented by Tempol, a superoxide dismutase mimetic. The decline in maximum force with Sin-1 and ONOO− treatments could be partially reversed by DTT, provided it was applied before the fiber was activated. Reversal by DTT indicates that the decrease in maximum force was due at least in part to oxidation of cysteine residues. Ascorbate caused similar reversal, further suggesting that the cysteine residues had undergone S-nitrosylation. The reduction in Ca2+ sensitivity, however, was not reversed by either DTT or ascorbate. Western blot analysis showed cross-linking of myosin heavy chain (MHC) I, appearing as larger protein complexes after ONOO− exposure. The findings suggest that ONOO− initially decreases maximum force primarily by oxidation of cysteine residues on the myosin heads, and that the accompanying decrease in Ca2+ sensitivity is likely due to other, less reversible actions of hydroxyl or related radicals.