Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins that use NAD(+) as the ADP-ribose donor. The reaction catalyzed by ETA involves the nucleophilic attack of the diphthamide residue on the anomeric carbon of the nicotinamide ribose forming a new glycosidic bond. A fluorometric assay involving the use of etheno-beta-nicotinamide adenine dinucleotide (epsilon-NAD(+)), an analog of NAD(+), has been found to provide a rapid, reliable, and sensitive procedure for assessing the kinetic parameters of this class of enzymes including ETA and its C-terminal fragment, PE24. Furthermore, application of this new assay facilitated the determination of the kinetic parameters for the protein substrate of ETA, elongation factor, which has previously been difficult to characterize. These findings provide new insights into catalytic mechanism of dipthamide-specific ribosyltransferases. In addition, this assay should also prove valuable for the study of NADases or NAD(+)-glycohydrolase enzymes (B. Weng, W. C. Thompson, H. J. Kim, R. L. Levine, and J. Moss, 1999, J. Biol. Chem. 274, 31797-31803; Y. S. Cho, M. K. Han, O. S. Kwark, M. S. Phoe, Y. S. Cha, N. H. An, and U. H. Kim, 1998, Comp. Physiol. B: Biochem. Mol. Biol. 120, 175-181) and the poly-ADP-ribosyltransferases (A. A. Pieper, A. Verma, J. Zhang, S. H. Snyder, 1999, Trends Pharmacol. Sci. 20, 171-181; M. K. Jacobson and E. L. Jacobson, 1999, Trends Biochem. Sci. 24, 415-417).