The yeast Saccharomyces cerevisiae has a single actin gene, ACT1, whose protein product is essential for cell viability. To study the structure-function relationship of this evolutionarily highly conserved protein, we have introduced into the gene several mutations leading to substitutions of amino acids that, by chemical crosslinking experiments, have previously been identified as potential sites for the interaction of actin with several actin-binding proteins and of actin monomers in filaments. The in vitro mutated actin genes were used to replace one chromosomal ACT1 allele in diploid cells. From diploid transformants, haploids that solely depended on mutant actins were isolated and their phenotypic alterations studied. The replacement of the N-terminal acidic residues (Asp2 and Glu4) with valine was functionally neutral. Substitutions of Asp11 led to dominant lethality. Substitutions of Lys191, Lys336, Trp356, Lys373 and Cys374 were without observable effect on cell growth, proliferation and morphology. Deletion of the C-terminal end, Lys-Cys-Phe-COOH, was lethal, whereas successive removal of the C-terminal Phe375 or Cys374 and Phe375 resulted in temperature sensitivity. At the nonpermissive temperature, the mutant cells were characterized by an increase in size, a tendency to lyse and significant alterations of the actin cytoskeleton.