Bovine liver glutamate dehydrogenase has been reacted with trinitrobenzenesulfonate and the primary sites of modification identified as lysine-428 and lysine-425 in the tentative amino acid sequence proposed by Smith, E. L., Landon, M., Piszkiewicz, D., Brattin, W. J., Jr., Langley, T. J., and Melamed, M. D. (1970), Proc. Nat. Acad. Sci. U. S. 67, 724). Lysine-428 reacts most rapidly but only to the extent of 0.5 group/polypeptide chain (i.e., three of the six subunits in the active monomer are modified). The reaction at lysine-425 proceeds moee slowly but also reaches an end point of only 0.5 group/polypeptide chain. Examination of the rate and extent of incorporation of trinitrobenzenesulfonate into these residues indicates that lysine-425 may be essentially unavailable for modification until the available lysine residues as position 428 have been trinitrophenylated. Sequence analysit of the tryptic peptides containing these residues shows that the modifications at positions 428 and 425 occur on different polypeptide chains. There appears to be no polypeptide chain in which both these residues are modified. No difference in the extent of modification of lysyl residues 428 or 425 was observed when nucleotide ligands which protect against loss of catalytic activity during trinitrophenylation were added to the reaction mixture. Procedures for the purification of relatively large trinitrophenylated peptides on a preparative scale in good yields, which have been largely developed in these experiments, are described.