The conventional role of eukaryotic elongation factor 1A (eEF1A) is to transport aminoacyl tRNA to the A site of ribosomes during the peptide elongation phase of protein synthesis. eEF1A also is involved in regulating the dynamics of microtubules and actin filaments in cytoplasm. In Tetrahymena, eEF1A forms homodimers and bundles F-actin. Ca(2+)/calmodulin (CaM) causes reversion of the eEF1A dimer to the monomer, which loosens F-actin bundling, and then Ca(2+)/CaM/eEF1A monomer complexes dissociate from F-actin. eEF1A consists of three domains in all eukaryotic species, but the individual roles of the Tetrahymena eEF1A domains in bundling F-actin are unknown. In this study, we investigated the interaction of each domain with F-actin, recombinant Tetrahymena CaM, and eEF1A itself in vitro, using three glutathione-S-transferase-domain fusion proteins (GST-dm1, -2, and -3). We found that only GST-dm3 bound to F-actin and influences dimer formation, but that all three domains bound to Tetrahymena CaM in a Ca(2+)-dependent manner. The critical Ca(2+) concentration for binding among three domains of eEF1A and CaM were < or =100 nM for domain 1, 100 nM to 1 microM for domain 3, and >1 microM for domain 2, whereas stimulation of and subsequent Ca(2+) influx through Ca(2+) channels raise the cellular Ca(2+) concentration from the basal level of approximately 100 nM to approximately 10 microM, suggesting that domain 3 has a pivotal role in Ca(2+)/CaM regulation of eEF1A.