Abstract Using 1,2-cyclohexanedione, modification of three arginines per actin monomer in F-actin resulted in a loss of ability of the actin to interact with tropomyosin, although the F-actin polymer was not significantly depolymerized, the ability of the actin to activate the Mg 2+ -ATPase of myosin was not affected, and the secondary structure of the actin monomers was not appreciably altered. Isolation of peptides from a digest of modified F-actin indicated that the modified residues were Arg-28, Arg-95 and Arg-147. When actin was combined with tropomyosin prior to the modification treatment, Arg-95 was not modified, and the actin retained its ability to bind tropomyosin. These results therefore indicate a direct involvement of Arg-95 in the tropomyosin binding function of F-actin.