We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle enzymes and in particular on the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle, IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2 O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mito-chondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2 O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.