Skeletal muscle mitochondrial dysfunction occurs in many conditions including aging and insulin resistance, but the molecular pathways of the mitochondrial dysfunction remain unclear. Presently, no methodologies are available to measure synthesis rates of individual mitochondrial proteins, which limits our ability to fully understand the translational regulation of gene transcripts. Here, we report a methodology to measure synthesis rates of multiple muscle mitochondrial proteins, which, along with large-scale measurements of mitochondrial gene transcripts and protein concentrations, will enable us to determine whether mitochondrial alteration is due to transcriptional or translational changes. The methodology involves in vivo labeling of muscle proteins with l-[ring-(13)C(6)]phenylalanine, protein purification by two-dimensional gel electrophoresis of muscle mitochondrial fraction, and protein identification and stable isotope abundance measurements by tandem mass spectrometry. Synthesis rates of 68 mitochondrial and 23 nonmitochondrial proteins from skeletal muscle mitochondrial fraction showed a 10-fold range, with the lowest rate for a structural protein such as myosin heavy chain (0.16 +/- 0.04%/h) and the highest for a mitochondrial protein such as dihydrolipoamide branched chain transacylase E2 (1.5 +/- 0.42%/h). This method offers an opportunity to better define the translational regulation of proteins in skeletal muscle or other tissues.