The phospholipid cardiolipin (CL) is ubiquitous in eucaryotes and is unique in structure, subcellular localization, and potential function. Previous studies have shown that CL is associated with major respiratory complexes in the mitochondrial membrane. To determine whether CL biosynthesis requires the presence of intact respiratory complexes, we measured activity of CL synthase, which catalyzes the synthesis of CL from cytidine diphosphate diacylglycerol and phosphatidylglycerol, in Saccharomyces cerevisiae strains with genetic defects in the oxidative phosphorylation system. Assembly mutants of cytochrome oxidase had significantly reduced CL synthase activity, while assembly mutants of respiratory complex III and the F0F1-ATPase were less inhibited. To obtain further information on the activity of CL synthase, we purified the enzyme and compared the size of the catalytic protein with the functional molecular mass. The enzyme was solubilized by Triton X-100 from KSCN-extracted mitochondrial membranes of S. cerevisiae. The functional molecular mass of Triton-solubilized CL synthase, determined by radiation inactivation, was 150-240 kDa, indicating that the functional enzyme was a large complex. After partial purification, the enzyme eluted from a Superose 12 gel filtration column with an apparent molecular mass of 70 kDa. CL synthase was further purified by hydroxylapatite and cytidine diphosphate diacylglycerol affinity chromatographies, Mono Q anion exchange FPLC, and preparative gel electrophoresis. These steps led to identification of a 28-kDa protein, which had catalytic activity when eluted from an SDS-polyacrylamide gel. This 28-kDa protein also reacted with an antiserum that inactivated the enzyme. We conclude that yeast CL synthase is a 28-kDa protein, which forms an oligomeric complex whose biogenesis and/or activity is influenced by the assembly of cyto-chrome oxidase.