Peroxynitrite may be generated in and around muscles in several pathophysiological conditions (e.g., sepsis) and may induce muscle dysfunction in these disease states. The effect of peroxynitrite on muscle force generation has not been directly assessed. The purpose of the present study was to assess the effects of peroxynitrite administration on diaphragmatic force-generating capacity in 1) intact diaphragm muscle fiber bundles (to model the effects produced by exposure of muscles to extracellular peroxynitrite) and 2) single skinned diaphragm muscle fibers (to model the effects of intracellular peroxynitrite on contractile protein function) by examining the effects of both peroxynitrite and a peroxynitrite-generating solution, 3-morpholinosydnonimine, on force vs. pCa characteristics. In intact diaphragm preparations, peroxynitrite reduced diaphragm force generation and increased muscle levels of 4-hydroxynonenal (an index of lipid peroxidation). In skinned fibers, both peroxynitrite and 3-morpholinosydnonimine reduced maximum calcium-activated force. These data indicate that peroxynitrite is capable of producing significant diaphragmatic contractile dysfunction. We speculate that peroxynitrite-mediated alterations may be responsible for much of the muscle dysfunction seen in pathophysiological conditions such as sepsis.