Phospholipids and tightly bound cardiolipin (CL) can be removed from Tween 20 solubilized bovine cytochrome bc(1) (EC 220.127.116.11) by digestion with Crotalus atrox phospholipase A(2). The resulting CL-free enzyme exhibits all the spectral properties of native cytochrome bc(1), but is completely inactive. Full electron transfer activity is restored by exogenous cardiolipin added in the presence of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE), but not by cardiolipin alone or by mixtures of phospholipids lacking cardiolipin. Acidic, nonmitochondrial phospholipids, e.g., monolysocardiolipin or phosphatidylglycerol, partially reactivate CL-free cytochrome bc(1) if they are added together with DOPC and DOPE. Phospholipid removal from the Tween 20 solubilized enzyme, including the tightly bound cardiolipin, does not perturb the environment of either cytochrome b(562) or b(566), nor does it cause the autoreduction of cytochrome c(1). Cardiolipin-free cytochrome bc(1) also binds antimycin and myxothiazol normally with the expected red shifts in b(562) and b(566), respectively. However, the CL-free enzyme is much less stable than the lipid-rich preparation, i.e., (1) many chromatographic methods perturb both cytochrome b(566)() (manifested by a hypsochromic effect, i.e., blue shift of 1.5-1.7 nm) and cytochrome c(1) (evidenced by autoreduction in the absence of reducing agents); (2) affinity chromatographic purification of the enzyme causes pronounced loss of subunits VII and XI (65-80% decrease) and less significant loss of subunits I, IV, V, and X (20-30% decrease); and (3) high detergent-to-protein ratios result in disassembly of the complex. We conclude that the major role of the phospholipids surrounding cytochrome bc(1), especially cardiolipin, is to stabilize the quaternary structure. In addition, bound cardiolipin has an additional functional role in that it is essential for enzyme activity.