The budding yeast Saccharomyces cerevisiae is an important model organism to study cellular structure and function. Due to its excellent accessibility to genetics and biochemical and microscopic analyses, studies with yeast have provided fundamental insights into mitochondrial biology. Yeast offers additional advantages because it can grow under fermenting conditions when oxidative phosphorylation is not obligatory and because the majority of mitochondrial structure and function are largely conserved during evolution. Isolation of mitochondria is an important technique for mitochondrial studies. This chapter focuses on procedures for the isolation and purification of intact yeast mitochondria that can be used for numerous functional assays as well as for analyses of mitochondrial ultrastructure.