Publisher Summary Saccharomyces cerevisiae is a facultative aerobe that requires an external source of unsaturated fatty acid and sterol for anaerobic growth. These lipid requirements have been exploited in a number of membrane studies to manipulate the membrane lipid composition of anaerobically grown yeast cells. However, anaerobiosis directly affects the synthesis of many other systems, such as the mitochondrial cytochromes, and thus it is desirable to be able to manipulate the membrane lipid composition aerobically. To manipulate the mitochondrial membrane lipid composition under aerobic growth conditions, mutants of yeast, auxotrophic for a membrane lipid component, can be utilized. In these mutants, the membrane concentration of the auxotrophic requirement is dependent on the availability of such requirement in the growth media, and it is possible to modify the membrane lipid composition of the mutants by varying the composition of the growth media. The chapter shows that the lipid mutants of yeast include the unsaturated fatty acid-requiring mutants, and the palmitate and sterol auxotrophs.