Saccharomyces cerevisiae is able to switch from fermentation to respiration (diauxic shift) with major changes in metabolic activity. This phenomenon has been previously studied on the transcriptional level. Here we present a parallel analysis of the yeast mitochondrial proteome and the corresponding transcriptional activity in cells grown on glucose (fermentation) and glycerol (respiration). A two-dimensional reference gel for this organelle proteome was established (available at www.biochem.oulu.fi/proteomics/), which contains about 800 intense spots. From 459 spots 253 individual proteins were identified, among them low abundant and hydrophobic proteins, and 37 proteins previously deemed hypothetical, with partially unknown cellular localization. After the diauxic shift, mitochondrial levels of only 18 proteins were changed (17 increased, with 1 decreased), among them proteins involved in the tricarboxylic acid cycle (Sdh1p, Sdh2p, and Sdh4p) and the respiratory chain (Cox4p, Cyb2p, and Qcr7p), proteins contributing to other respiratory pathways (Ach1p, Adh2p, Ald4p, Cat2p, Icl2p, and Pdh1p), and two proteins with unknown function (Om45p and Ybr230p). Apart from an overall increase in mitochondrial protein mass, the mitochondrial proteome remains remarkably constant, even in a major metabolic adaptation. This seemingly disagrees with results of the DNA microarray analyses, where a rather heterogenous up- or down-regulation of genes encoding mitochondrial proteins implies large changes in the proteome. We propose that the discrepancy between proteome and transcriptional regulation, apart from different translation efficiency, indicates a changed turnover rate of proteins in different physiological conditions.