In eukaryotic cells, the mitochondrion is the key organelle for cellular respiration. Mitochondrial proteome analysis is difficult to perform by the classical proteomic approach involving two-dimensional gel electrophoresis (2DE), because this organelle contains a large number of membrane-associated and highly alkaline proteins usually requiring specific treatments to be successfully analyzed. Here, an alternative approach was evaluated and led to the rapid and sensitive identification of approximately 35% of the yeast mitochondrial proteins. It consists of an SDS-PAGE gel electrophoresis of the total mitochondrial protein in combination with the LC-MS/MS analysis of the digestion products of gel slices. The use of only 40 microg of mitochondrial protein enabled the identification of 179 different gene products divided into similar proportions of membrane and soluble proteins. The distribution of the identified proteins in terms of pI and hydrophobicity revealed that the present analytical strategy is largely unbiased. The identification of 28 proteins of previously unknown subcellular localization demonstrated the ability of SDS-PAGE-LC-MS/MS to rapidly supplement the knowledge of the mitochondrial proteome.