The identification of a majority of the polypeptides in mitochondria would be invaluable because they play crucial and diverse roles in many cellular processes and diseases. The endogenous production of reactive oxygen species (ROS) is a major limiter of life as illustrated by studies in which the transgenic overexpression in invertebrates of catalytic antioxidant enzymes results in increased lifespans. Mitochondria have received considerable attention as a principal source—and target—of ROS. Mitochondrial oxidative stress has been implicated in heart disease including myocardial preconditioning, ischemia/reperfusion, and other pathologies. In addition, oxidative stress in the mitochondria is associated with the pathogenesis of Alzheimer’s disease, Parkinson’s disease, prion diseases, and amyotrophic lateral sclerosis (ALS) as well as aging itself. The rapidly emerging field of proteomics can provide powerful strategies for the characterization of mitochondrial proteins. Current approaches to mitochondrial proteomics include the creation of detailed catalogues of the protein components in a single sample or the identification of differentially expressed proteins in diseased or physiologically altered samples versus a reference control. It is clear that for any proteomics approach prefractionation of complex protein mixtures is essential to facilitate the identification of low-abundance proteins because the dynamic range of protein abundance within cells has been estimated to be as high as 107. The opportunities for identification of proteins directly involved in diseases associated with or caused by mitochondrial dysfunction are compelling. Future efforts will focus on linking genomic array information to actual protein levels in mitochondria.