A mAb (1E5) that binds the COOH-terminal region of the β subunit of chicken CapZ inhibits the ability of CapZ to bind the barbed ends of actin filaments and nucleate actin polymerization. CapZ prepared as fusion proteins in bacteria or nonfusion proteins by in vitro translation has activity similar to that of CapZ purified from muscle. Deletion of the COOH-terminus of the β subunit of CapZ leads to a loss of CapZ's ability to bind the barbed ends of actin filaments. A peptide corresponding to the COOH-terminal region of CapZ β, expressed as a fusion protein, binds actin monomers. The mAb 1E5 also inhibits the binding of this peptide to actin. These results suggest that the COOH-terminal region of the β subunit of CapZ is an actin-binding site. The primary structure of this region is not similar to that of potential actin-binding sites identified in other proteins. In addition, the primary structure of this region is not conserved across species.