Quantitative determination of cardiolipin from two mitochondrial electron-transferring complexes was achieved using a rapid and sensitive silicic acid HPLC method combined with digital analysis of the elution profile. Phospholipid samples containing as little as 0. 01 nmol of cardiolipin were accurately analyzed. Phospholipids from detergent-solubilized cytochrome bc1 (EC 1.10.2.2) and cytochrome c oxidase (EC 1.9.3.1) were extracted by an organic two-phase system and analyzed by isocratic normal-phase HPLC after dissolving the dried sample in the mobile phase (cyclohexane:2-propanol:5 mM phosphoric acid, 50:50:2.9, v/v/v). Analysis was performed by the method of standard addition in which increasing amounts of cardiolipin (0 to 5 nmol) are added to a constant amount of phospholipid extract containing an unknown amount of cardiolipin. By determining the slope and intercept of a plot of the HPLC elution peak area as a function of the amount of standard cardiolipin added, the amount of cardiolipin in the unknown is determined. By this analysis, purified, detergent-solubilized bovine heart cytochrome bc1 and cytochrome c oxidase contained 9.2 +/- 0.7 and 3.05 +/- 0.05 mol cardiolipin per mole of enzyme, respectively. The method was also used to prove that cardiolipin could be completely removed from each complex by digestion with Crotalus atrox phospholipase A2, i.e., each delipidated complex contained less than 0.05 mol cardiolipin per mole of complex. The rapidity and high sensitivity of this method make it very useful for analysis of cardiolipin in other biological samples.