Phosphatidylglycerophosphate (PGP) synthase catalyzes a reaction involved in the synthesis of phosphatidylglycerol (PG), which serves as a metabolic precursor for cardiolipin (CL), found primarily in the mitochondrial membranes of eukaryotic cells. We isolated a Chinese hamster ovary cell mutant (designated PGS-S) with a specific lesion in PGP synthase by using an in situ enzymatic assay for the enzyme. This mutant was obtained by introducing a second mutation into mutant PGS-P that had been generated by first-step mutagenesis. The PGP synthase activities in cell extracts of mutant PGS-S grown at 33 and 40 degrees C were 14 and 1% of those in the wild type cells, respectively; in addition, PGP synthase in cell extracts of mutant PGS-S exhibited higher sensitivity to heat than that of the wild type. Mutant PGS-S also showed a temperature-dependent defect in the synthesis of PG and CL in vivo, together with temperature sensitivity for cell growth. A temperature-resistant revertant of mutant PGS-S simultaneously restored PGP synthase activity and the ability to synthesize PG and CL in vivo to nearly the same levels as those of mutant PGS-P. These results constitute genetic evidence that PGP synthase is responsible for PG synthesis and is essential for cell growth.