A DNA fragment from yeast (Saccharomyces cerevisiae) was identified by its homology to a chicken beta-tubulin cDNA and cloned. The fragment was shown to be unique in the yeast genome and to contain the gene for yeast beta-tubulin, since it can complement a benomyl-resistant conditional-lethal mutation. A smaller subfragment, when used to direct integration of a plasmid to the benomyl resistance locus in a diploid cell, disrupted one of the beta-tubulin genes and concomitantly created a recessive lethal mutation, indicating that the single beta-tubulin gene of yeast has an essential function. Determination of the nucleotide sequence reveals extensive amino acid sequence homology (more than 70%) between yeast and chicken brain beta-tubulins.