Two derivatives of hen egg-white lysozyme (lysozyme) with single substitutions of a 2,4-dinitrophenyl (DNP) residue were prepared. The reaction of lysozyme with a 10-fold molar excess of 2,4-dinitrobenzene sulfonic acid provided one mono-DNP substituted lysozyme (DNP₁-33lysozyme), which was purified by ion-exchange chromatographies. The other one (DNP₁-96lysozyme) was prepared as follows. After maleylation of lysozyme in the presence of a 7-fold molar excess of maleic anhydride, the derivative with one free amino group was purified on DE-52. This material was dinitrophenylated with 2,4-dinitrobenzene sulfonic acid and the mono-DNP substituted derivative was purified on DE-52. DNP₁-96lysozyme was finally purified on SE-Sephadex C-25, after demaleylation at pH 3.5, at 37°C, for 5 days. DNP₁-33lysozyme and DNP₁-96lysozyme each migrated as a single band with slower mobility than that of native lysozyme. On reduction, carboxymethylation and chymotrypsin digestion, both mono-DNP substituted lysozymes yielded a single yellow peptide. The amino acid compositions or partial sequence of these peptides indicated that lysine-33 and lysine-96 were the only dinitrophenylated residues in DNP₁-33lysozyme and DNP₁-96lysozyme, respectively. DNP₁-33lysozyme and DNP₁-96lysozyme showed antigenic reactivities equal to that of native lysozyme with antisera to lysozyme. The DNP residues on the protein were accessible to anti-DNP antibodies, but the affinities of DNP₁-33lysozyme to anti-DNP antibodies were lower than those of DNP₁-96lysozyme. This result is discussed with respect to the local environments of the DNP residues in these proteins.