Elongation factor-3 (EF-3) is an essential translation elongation factor specific to yeasts and fungi. When EF-3 interacts with yeast ribosomes, its ATPase activity, which is indispensable for the function of EF-3 in translation, is drastically enhanced. In this study, a monoclonal antibody specific for the carboxy-terminal region inhibited the ribosome-activated ATPase activity of EF-3, while it did not inhibit the intrinsic ATPase activity of EF-3. The results suggest that the carboxy-terminal region of EF-3 is involved in the interaction with yeast ribosomes. The monoclonal antibody also inhibited poly(U)-directed poly(Phe) synthesis, which indicates that the carboxy-terminal region is important for EF-3 to express its function in the polypeptide elongation cycle.