Publisher Summary This chapter discusses the isolation of protein import components from saccharomyces cerevisiae mitochondria and some biochemical techniques that have been used by the laboratory for the identification and characterization of several of these import components from yeast mitochondria. Antibodies have proven to be extremely useful for the identification and characterization of several new import components. For example, when multivalent antibodies were added to right-side-out inner membrane vesicles, protein import was inhibited. The antigen recognized by the inhibitory antibody could then be traced and characterized. Such studies require careful choice of the solubilization conditions. A mild, nonionic detergent is recommended to avoid the disruption of protein–protein interactions. Because the choice of detergent is empirical, several detergents should always be tested to determine the optimal conditions. The salt concentration may also have an effect and should be optimized. In some cases, it may be useful to add glycerol or sucrose to stabilize protein–protein interactions.