Translation elongation factor 1α (EF-1α) catalyzes the GTP-dependent binding of aminoacyl-tRNA to the ribosome. Previously, Tetrahymena 14-nm filament-associated protein was identified as EF-1α [Kurasawa et al. (1992) Exp. Cell Res. 203, 251-258]. This and several other studies suggest that EF-1α functions not only in translation but also in regulation of some part of the cytoskeleton. Tetrahymena EF-1α bound to F-actin and induced bundling of F-actin. We investigated the effects of GTP/GDP and Ca²⁺/calmodulin on F-actin bundling activity of EF-1α. The presence of GTP, GDP, or guanylyl-imidodiphosphate (GMP-PNP) slightly decreased the amount of EF-1α which bound to F-actin, but each had virtually no effect on the F-actin bundling activity. The formation of F-actin bundles by EF-1α was Ca²⁺-insensitive. In the absence of Ca²⁺, calmodulin did not bind to EF-1α and F-actin. On the other hand, in the presence of Ca²⁺, calmodulin directly bound to EF-1α but did not have any serious influence on EF-1α/F-actin binding. Under the conditions, electron microscopy demonstrated that Ca²⁺/calmodulin completely inhibited the F-actin bundling by EF-1α. These results indicate that Ca²⁺/calmodulin regulates the F-actin bundling activity of EF-1α without inhibition of the binding between EF-lα and F-actin.