To further elucidate the functions of actin in budding yeast and to relate actin structure to specific roles and interactions in vivo, we determined the phenotypes caused by 13 charged-to-alanine mutations isolated previously in the single Saccharomyces cerevisiae actin gene. Defects in actin organization, morphogenesis, budding pattern, chitin deposition, septation, nuclear segregation, and mitochondrial organization were observed. In wild-type cells, mitochondria were found to be aligned along actin cables. Many of the amino acid substitutions that had the most severe effects on mitochondrial organization are located under the myosin "footprint" on the actin monomer, suggesting that actin-myosin interactions might underlie mitochondrial organization in yeast. In addition, one mutant (act1-129; R177A, D179A) produced an actin that assembled into cables and patches that could be visualized by anti-actin immunofluorescence in situ and that assembled into microfilaments of normal appearance in vitro as judged by electron microscopy but which could not be labeled by rhodamine-phalloidin in situ or in vitro. Rhodamine-phalloidin could label actin filaments assembled from all of the other mutant actins, including one (act1-119; R116A, E117A, K118A) that is altered at a residue (E117) that can be chemically cross-linked to phalloidin. The implication of residues R177 and/or D179 in phalloidin binding is in close agreement with a recently reported molecular model in which the phalloidin-binding site is proposed to be at the junction of two or three actin monomers in the filament.