Characterizing mutants of actin that do not polymerize will advance our understanding of the mechanism of actin polymerization and will be invaluable for the production of short F-actin structures for structural studies. To circumvent the problem of expressing dominant lethal nonpolymerizing actin in yeast, we adopted a cysteine engineering strategy. Here we report the characterization of a mutant of yeast actin, AC-actin, possessing a single pointed-end mutation, A204C. Expression of this mutant in yeast results in actin-polymerization-deficient phenotypes. When copolymerized with wild-type actin, ATP-AC-actin is incorporated into filaments. ADP-AC-actin participates in the nucleation and elongation of wild-type filaments only at very high concentrations. At low concentrations, ADP-AC-actin appears to participate only in the nucleation of wild-type filaments, suggesting that Ala-204 is involved in modulating the critical concentration of the pointed end of actin.