Actin, a major cytoskeletal component of all eukaryotic cells, is one of the most highly conserved proteins. It is involved in various cellular processes such as motility, cytoplasmic streaming, chromosome segregation and cytokinesis. The actin from the yeast Saccharomyces cerevisiae, encoded by the essential ACT1 gene, is 89% identical to mouse cytoplasmic actin and is involved in the organization and polarized growth of the cell surface. We report here the characterization of ACT2, a previously undescribed yeast split gene encoding a putative protein (391 amino acids, relative molecular mass (Mr) 44,073) that is 47% identical to yeast actin. The requirement of the ACT2 gene for vegetative growth of yeast cells and the existence of related genes in other eukaryotes indicate an important and conserved role for these actin-like proteins. Superimposition of the Act2 polypeptide onto the three-dimensional structure of known actins reveals that most of the divergence occurred in loops involved in actin polymerization, DNase I and myosin binding, leaving the core domain mainly unaffected. To our knowledge, the Act2 protein from S. cerevisiae is the first highly divergent actin molecule described. Structural and physiological data suggest that the Act2 protein might have an important role in cytoskeletal reorganization during the cell cycle.