Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning. Antibodies that recognized mitochondrial proteins were identified by screening human liver cDNA expression libraries. In this study, we identified 6 major antigens that were recognized by at least 2 different monoclonal antibodies (mAbs). The proteins that were antigenically dominant were: acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase), aldehyde dehydrogenase 1 family member A1, carbamoyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), enoyl coenzyme A hydratase 1, and hydroxysteroid (11-beta) dehydrogenase 1. We also determined the subcellular localizations of these enzymes within the mitochondria using immunohistocytochemistry. We believe that these well-characterized antibodies will provide a valuable resource for the Human Liver Proteome Project (HLPP), and will make studies aimed at investigating liver mitochondrial function far easier to perform in future. Our results provide strong evidence that, (i) depletion of dominant proteins from liver mitochondrial samples is possible and, (ii) the approaches adopted in this study can be used to explore or validate protein-protein interactions in this important organelle.