Although protein carbonyl formation is an index of oxidative stress in skeletal muscles, the exact proteins, which undergo oxidation in these muscles, remain unknown. We used 2D electrophoresis, immunoblotting, and mass spectrometry to identify carbonylated proteins in the diaphragm in septic animals. Rats were injected with saline (control) or Escherichia coli lipopolysaccharides (LPS) and killed after various intervals. Diaphragm protein carbonylation increased significantly and peaked 12 h after LPS injection, and it was localized both inside muscle fibers and in blood vessels supplying muscle fibers. Aldolase A, glyceraldehyde 3-phosphate dehydrogenase, enolase 3beta, mitochondrial and cytosolic creatine kinases, alpha-actin, carbonic anyhdrase III, and ubiquinol-cytochrome c reductase were all carbonylated in septic rat diaphragms. In addition, we found significant negative correlations between the intensity of carbonylation and creatine kinase and aldolase activities. We conclude that glycolysis, ATP production, CO2 hydration, and contractile proteins are targeted by oxygen radicals inside the diaphragm during sepsis.