Affinity purification of the membrane-bound, respiratory chain protein cytochrome c oxidase (COX) from Rhodobacter sphaeroides yields an enzyme with an average of 5 phospholipids per mole enzyme as measured using inductively coupled plasma-mass spectrometry. The crystal structure of COX shows two phospholipids within subunit III and four at the interface between subunits I, III and IV. Phospholipase A2 treatment of COX for 3 hours at 4 °C in 20 mM MOPS pH 7.2, 20 mM CaCl2 and 0.2 % dodecyl maltoside removes phospholipid (< 1 mole phospholipid per mole enzyme) from COX and results in a 30% decrease in electron transfer activity. Lipid-depleted COX also exhibits the phenomenon of turnover-induced inactivation or suicide inactivation. Inhibition of electron transfer activity and suicide inactivation were reversed by the addition of 1 mg/ml soya bean asolectin (a mixture of phospholipids). 0.5 mM cardiolipin [CL](C18:1 fatty acid chain length: double bond) or 0.5 mM fatty acids with side chains greater than 12 carbon atoms including arachadonic acid also reconstituted the activities in the lipid-depleted enzyme, whereas phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl glycerol, and phosphatidyl choline (all with C18:1 side chains) used at similar concentrations did not. Lipid-depleted COX after incubation with CL exhibited a shift in migration on blue native gel electrophoresis at pH 7.0, suggesting that CL binding modifies the conformation of COX. To assess protein flexibility upon removal of lipid, both control and lipid-depleted COX were labeled in subunit III with the fluorophore, N-iodoacetylamindoethyl-1-aminonaphthalene-5-sulfonate (IAEDANS). Fluorescence anisotropy measurements showed a 50% increase in the rotational rate of AEDANS-labeled lipid-depleted COX. This increase in flexibility of subunit III upon lipid removal translates to the adjacent subunit I which contains the catalytic center. These results taken together suggest that CL binds to COX to stabilize the conformation of the enzyme for maximum catalytic efficiency.