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 whether 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 nitrosoglutathione (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, whereas 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 were retained. Interestingly, neither molecule had any effect on the Ca2+ sensitivity of slow-twitch skeletal muscle. The data show that H2O2 and GSNO retain the capacity to independently affect the contractile apparatus to modulate force. Furthermore, the absence of effects in slow-twitch muscle may further explain why this fiber type is relatively insensitive to fatigue.