The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth. Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere [Buzan, J., Du, J., Karpova, T., and Frieden, C. (1999) Proc. Natl, Acad, Sci, USA 96, 2823-2827]. The mutations, in muscle act in numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament. The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth. The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca. 4 mu M) without a large change in the ability to form nuclei for the nucleation-elongation process.