Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH.