Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains the binding sites for anionic phospholipids, heparin, and F-actin, whereas the p11 subunit provides a regulatory function. The F-actin-binding site is presently unknown. In the present study we have utilized site-directed mutagenesis to create annexin II mutants with truncations in the C terminus of the molecule. Interestingly, a mutant annexin II lacking its C-terminal 16, 13, or 9 amino acids was unable to bind to F-actin but still retained its ability to interact with both anionic phospholipids and heparin. Recombinant AIIt, composed of wild-type p11 subunits and the mutant annexin II subunits, was also unable to bundle F-actin. This loss of F-actin bundling activity was directly attributable to the inability of mutant AIIt to bind F-actin. These results establish for the first time that the annexin II C-terminal amino acid residues, LLYLCGGDD, participate in F-actin binding.