Eukaryotic elongation factor 2 (eEF2) contains a post-translationally modified histidine residue, referred to as diphthamide, which is the specific ribosylation target of exotoxin A (ETA) secreted by 'Pseudomonas aeruginosa. ' In yeast eEF2, the diphthamide-containing loop (Leu-693-Gly-703) is located at the tip of domain IV. To elucidate the role of the diphthamide-containing loop in the ADP-ribosylation of eEF2, the conserved residues within this loop were replaced with Ala through the use of site-directed mutagenesis. The structural integrity and ADP-ribose acceptor activities of the purified mutant eEF2 proteins were characterized by a variety of fluorescence-based approaches. The single-Ala mutations in the diphthamide-containing loop produced eEF2 proteins with extremely low ADP-ribose acceptor activities, which indicated that this loop plays an important role in the ADP-ribosylation catalyzed by ETA. In addition, the naked imidazole ring of Diph-699 is essential for the acceptance of the ADP-ribose moiety during the toxin-catalyzed reaction.