When bovine cytochrome c was trinitrophenylated with trinitrobenzenesulfonate (TNBS) under the specific conditions, the trinitrophenylated (TNP-) cytochrome c preparations, in which 1- to 5-TNP-groups were incorporated into the ε-amino groups of lysine residues, were obtained separately in homogeneous state. Several properties of the TNP-cytochrome c were examined as compared with those of the acetylated ( 1) and native cytochrome c. It was found that the activity of TNP-cytochrome c in the cytochrome oxidase [EC 1.9.3. 1] reaction was less than a half of that of native one, even when only one TNP-group was incorporated into cytochrome c molecule. From the amino acid analyses of these preparations of TNP-cytochrome c, it was found that the 13th lysine residue from the amino terminus of the protein was most reactive with TNBS and the 22nd lysine residue was secondly reactive. The remarkable loss in activity of TNP-cytochrome c in the cytochrome oxidase reaction was discussed as compared with the case of acetyl-cytochrome c.