Publisher Summary This chapter discusses the chemical modification of mitochondria by cross-linking agents. Bifunctional alkylating agents or cross-linking agents can be used to investigate lateral associations among membrane proteins. Normally, these noncovalent associations are destroyed when membranes are solubilized and analyzed. Cross-linking of neighboring proteins prevents dissociation of apposed proteins allows for subsequent analysis by techniques, such as gel filtration or electrophoresis. Because cross-linking would be expected to reduce the mobility of membrane components, treated membranes can be used as model systems to investigate the role of molecular motion in membrane function. The chapter reviews the number of compounds have been used to crosslink membrane proteins. However, bifunctional imidoesters appear to be particularly advantageous as crosslinking agents. For example, these reagents exist as monomers in aqueous solutions and are available in various chain lengths. This latter property makes possible the use of biimidates as molecular rulers to determine distances among membrane components. Imidates react primarily with free amines to yield cationic amidines. This conservation of charge is apparently responsible for the reported mildness of the amidination reaction.