Tetrahymena F-actin-binding protein, which induces bundling of Tetrahymena F-actin, was localized to a division furrow during cytokinesis. We report here the cloning and characterization of the gene and cDNA of a Tetrahymena F-actin-binding protein. The cDNA encodes a protein comprising 579 deduced amino acids with a calculated molecular mass of 65.9 kDa. The predicted amino acid sequence shares 37.7, 41.8, and 39% identity with the sequences of yeast fimbrin, Arabidopsis thaliana fimbrin, and Dictyostelium discoideum plastin, respectively. The Tetrahymena F-actin-binding protein also shares two actin-binding domains previously identified in the fimbrin/plastin family, but lacks the EF-hand Ca²⁺-binding motif, suggesting that this protein is a novel-fimbrin-like protein in Tetrahymena. Moreover, we cloned a genomic DNA encoding the Tetrahymena fimbrin-like protein and performed Southern and Northern hybridizations. The results indicate that the genomic DNA possesses 9 introns and that both the gene and transcript of Tetrahymena fimbrin-like protein are single. Thus, we suggest that Tetrahymena fimbrin-like protein localizes to the division furrow and probably cross-links actin filaments in a Ca²⁺-insensitive manner during cytokinesis.