A systematic strategy was used to create a synoptic set of mutations that are distributed throughout the single 0-tubulin gene of Saccharomyces cerevisiae. Clusters of charged amino acids were targeted for mutagenesis and converted to alanine to maximize alterations on the protein's surface and minimize alterations that affect protein folding. Of the 55 mutations we constructed, three confer dominant-lethality, 11 confer recessive-lethality, 10 confer cold-sensitivity, one confers heat-sensitivity, and 27 confer altered resistance to benomyl. Only 11 alleles give no discernible phenotype. In spite of the fact that f-tubulin is a highly conserved protein, three-fourths of the mutations do not destroy the ability of the protein to support the growth of yeast at 30°C. The lethal substitutions are primarily located in three regions of the protein and presumably identify domains most critical for 0-tubulin function. Interestingly, most of the conditional-lethal alleles produce specific defects in spindle assembly at their restrictive temperature; cytoplasmic microtubules are relatively unaffected. The exceptions are two mutants that contain abnormally long cytoplasmic microtubules. Mutants with specific spindle defects were not observed in our previous collection of f-tubulin mutants and should be valuable in dissecting spindle function.