The reaction of lysozyme with ethylenimine has been studied at neutral and acidic pH. At least four singly modified lysozyme derivatives have been isolated. From the product analyses, only carboxyl groups in the protein were found to react. One of the derivatives, 1, was very labile and reverted back to native lysozyme during the isolation procedure. Its formation was markedly inhibited by the presence of tri(N-acetyl-D-glucosamine), indicating that the modified carboxyl residue in 1 is at or close to the binding site of lysozyme for the trisaccharide. Two other derivatives were identified as 2-aminoethyl esters of Glu-35 (2) and Asp-52 (3). At pH 10 and room temperature, these derivatives rearranged to the corresponding 2-hydroxyethylamide derivatives (5 and 6). A fourth derivative (4) did not contain an ethanolamine moiety; nevertheless, its Glu-35 was found to be modified by sequence analysis. Lysozyme derivatives modified at Glu-35 and Asp-52 were inactive toward glycol chitin but retained high affinity for tri(N-acetyl-D-glucosamine). Thus, Glu-35 and Asp-52 are essential for enzyme activity. A mechanism for the selective modification of these carboxyl residues in lysozyme has been proposed and related to the metal binding ability of lysozyme.