Almost complete phospholipid depletion has been achieved for Complex I and III of the mitochondrial respiratory chain using a technique that involves elution on Sephadex LH-20 in the presence of Triton X-100. Enzymic activity may be regenerated by replenishment with phospholipid. However, restoration of enzymic activity in phospholipid-depleted Complex I and III has been shown to require the presence of cardiolipin. These results are, therefore, similar to findings on the absolute catalytic requirement of cardiolipin for cytochrome oxidase activity (Fry, M., and Green, D. E. (1980) Biochem. Biophys. Res. Commun. 93, 1238-1246). At least two roles for phospholipid involvement in electron transfer processes are proposed, a catalytic role provided specifically by cardiolipin and a dispersive role that may be provided by various phospholipids or detergents. The absolute requirement of enzymic activity for cardiolipin suggests that this phospholipid plays a crucial role in the coupled electron transfer process.