Tryptophan residues of diphtheria toxin fragment A have been modified by 2-hydroxy-5-nitrobenzyl bromide. Fragment A loses its ability to inactivate ADP-ribosylation of the elongation factor 2, as a function of the number of residues modified. Modification of one tryptophan residue provides a dramatic loss of enzymic activity suggesting the presence of one essential residue of this type in fragment A. Examination of fingerprint maps of chymotryptic peptides of fragment A after reaction with 2-hydroxy-5-nitrobenzyl bromide allows us to identify this tryptophan residue at position 153 of the amino acid sequence. ORD and CD experiments joined to immunochemical studies seem to exclude the possibility that the observed decrease in the enzymic activity may be attributed to a conformational change. Finally, results of nicotinamide-adenine dinucleotide-binding measurements suggest that tryptophan 153 would be concerned with the elongation factor 2 binding site or with the catalytic site itself.