Modification of biomolecules by reactive aldehydes is believed to play a role in biological processes, including aging, atherosclerosis, and Alzheimer's disease. Here, the modification of cytochrome c promoted by trans, trans-2,4-decadienal (DDE) was investigated. Matrix-assisted laser desorption/ionization time-of-flight experiments indicated increases in the molecular weight of cytochrome c, consistent with the formation of DDE adducts. Our data show that the protein modification was time-, pH-, and DDE concentration-dependent, leading to the formation of at least six adducts after 2 h of incubation at pH 7.4. Electrospray ionization quantitative TOF mass spectrometry analysis of tryptic digests indicated that His-33, Lys-39, Lys-72, and Lys-100 were modified by DDE. These adducts could have significant effects considering that His-33, Lys-72, and Lys-100 are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with cardiolipin in the inner mitochondrial membrane and cytochrome c oxidase. A blue shift in the cytochrome c Soret band from 409 to 406 nm was also observed after DDE reaction, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. The covalent modifications in cytochrome c could play a role in mitochondrial dysfunction associated with oxidative stress.