Three steps of chromatography of a post-ribosomal supernatant fraction have provided a highly purified preparation of peptide elongation factor 3 (EF-3) with a molecular weight of 125,000 from the typical budding yeast Saccharomyces carlsbergensis and of the factor with a molecular weight of 120,000 from the fission yeast Schizosaccharomyces pombe. Both of the proteins consist of a single peptide chain. The purified factors fulfilled the requirement for polyphenylalanine synthesis on yeast ribosomes and exhibited strong ATPase and GTPase activities dependent on yeast ribosomes. The activity profiles of the nucleotidases dependent on pH and salt concentration and the inhibition studies indicated that the ATPase and GTPase activities of EF-3 were displayed by the same active site with a wide substrate specificity, showing the highest activity with ATP. Those experiments also revealed that the ATPase and GTPase of EF-3 were characteristically different from the GTPases of EF-1 alpha and EF-2. Both Km and kcat of EF-3 for ATP (Km = 0.12 mM and Kcat = 610 mol/mol/min) and GTP (Km = 0.20 mM and kcat = 390 mol/mol/min) are much higher than those of the GTPases of EF-1 alpha and EF-2. Inactivation experiments and studies on the ATP effect led us to conclude that this ATPase activity was an essential requirement for the functional role of EF-3 and therefore, in addition to the GTPases of EF-1 alpha and EF-2, the third nucleoside triphosphate hydrolyzing step by the ATPase of EF-3 was necessary for the yeast peptide elongation cycle.