Eukaryotic elongation factor 2 (EF-2) was shown to bind to F-actin as assayed by co-sedimentation. In the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding was increased fourfold. At saturation level a molar ratio of about 0.12 EF-2 per F-actin (subunit) was observed. Our results suggest a single type of binding site with an apparent dissociation constant of 0.85 microM. The stoichiometry was independent of the filament length, and ADP-ribosylation had no effect on the binding. Experimental data indicated the involvement of SH-groups of both EF-2 and actin in the binding. The interaction EF-2 with F-actin appeared to be inhibited competitively by EF-1 alpha and non-competitively by G-actin.