Reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and nitric oxide (NO), have been shown to differentially alter the Ca²⁺-sensitivity of the contractile apparatus of fast-twitch skeletal muscle leading to the proposal that normal muscle function is controlled by perturbations in the amounts of these two groups of molecules (28). However, no previous studies have examined if these opposing actions are retained when the contractile apparatus is subjected to both molecule types. Using mechanically skinned fast- and slow-twitch skeletal muscle fibers of the rat, we compared the effects of sequential addition of nitrosylglutathione (GSNO), a NO donor and H₂O₂ on the Ca₂₊-sensitivity of the contractile apparatus. As expected from previous reports in fast- twitch fibers when added separately GSNO (1 mM) reduced the Ca²⁺-sensitivity of the contractile apparatus while H₂O₂ (10 mM; added during contractions) increased the Ca²⁺-sensitivity of the contractile apparatus. When added sequentially to the same fiber, such that the oxidation by one molecule (e.g. GSNO) preceded the oxidation by the other (e.g. H₂O₂), and vice versa, the individual effects of both molecules on the Ca²⁺-sensitivity where retained. Interestingly, neither molecule had any effect on the Ca2+-sensitivity of slow-twitch skeletal muscle. These data show that the ROS, H₂O₂, and GSNO retain the capacity to independently affect the contractile apparatus to modulate force. Furthermore, the absence effects in slow-twitch muscle may further explain why this fiber type is relatively insensitive to fatigue.