The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besides its well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bind to eEF-2 [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D., and Reboud, J. P. (1993) Biochemistry 32, 1976-1980]. However, whether the adenylic and guanylic nucleotide binding sites were different or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presence of N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to an increase in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. The Mant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with a similar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggested the presence of two different sites binding either guanylic or adenylic nucleotides. A Förster's transfer between tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylic Mant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second binding site specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different species suggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s from higher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and located in this insert has appeared in the course of evolution although its physiological function is still unknown.