Microtubules play essential roles in a wide variety of cellular processes including cell division, motility, and vesicular transport. Microtubule function depends on the polymerization dynamics of tubulin and specific interactions between tubulin and diverse microtubule-associated proteins. To date, investigation of the structural and functional properties of tubulin and tubulin mutants has been limited by the inability to obtain functional protein from overexpression systems, and by the heterogeneous mixture of tubulin isotypes typically isolated from higher eukaryotes. The budding yeast, Saccharomyces cerevisiae, has emerged as a leading system for tubulin structure-function analysis. Yeast cells encode a single beta-tubulin gene and can be engineered to express just one of two alpha isotypes. Moreover, yeast allows site-directed modification of tubulin genes at the endogenous loci expressed under the native promoter and regulatory elements. These advantageous features provide a homogeneous and controlled environment for analysis of the functional consequences of specific mutations. Here, we present the techniques to generate site-specific tubulin mutations in diploid and haploid cells, assess the ability of the mutated protein to support cell viability, measure overall microtubule stability, and define changes in the specific parameters of microtubule dynamic instability. We also outline strategies to determine whether mutations disrupt interactions with microtubule-associated proteins. Microtubule-based functions in yeast are well defined, which allows the observed changes in microtubule properties to be related to the role of microtubules in specific cellular processes.