The elongation factors (EF-Tu/EF-1α) are universal proteins, involved in protein biosynthesis. A detailed characterization of the stability against temperature of SsEF-1α, a three-domain protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus is presented. Thermal denaturation of both the GDP-bound (SsEF-1α•GDP) and the ligand-free (nfSsEF-1α) forms was investigated by means of circular dichroism and fluorescence measurements, over the 4.0–7.5 pH interval. Data indicate that the unfolding process is cooperative with no intermediate species and that the few inter-domain contacts identified in the crystal structure of SsEF-1α play a role also at high temperatures. Finally, it is shown that the enzyme exhibits two different interchangeable thermally denatured states, depending on pH.