Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal β-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-offlight/ time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor α-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibratespecific induction scheme showing the variability of peroxisome proliferator-activated receptor α-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22-C26) but rather metabolize preferentially long chain fatty acids (C16-18). © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.