Tetrahymena 14-nm filament protein/citrate synthase (49K protein) is a bifunctional protein with roles in the cytoskeleton and as a citrate synthase. Though previous studies have shown that the 49K protein is derived from a single transcript of a single gene, direct demonstration of the 49K protein's bifunctional property remained to be elucidated. In this study, a recombinant 49K protein was expressed in Escherichia coli, purified and characterized. The citrate synthase activity of the recombinant 49K protein was comparable to that of the 49K protein purified from Tetrahymena. The recombinant 49K protein formed 14-nm filaments, but only of short length. The filaments were elongated in the presence of a soluble fraction of Tetrahymena. These results suggest that the 49K protein itself is bifunctional, but some co-factor(s) is necessary for elongation of filaments.