The conversion of phosphatidylglycerol(PG) to cardiolipin(CL) in isolated Staphylococcal cell membrane was studied to clarify the physiological role of the reaction in the membrane. CL content in the isolated membrane was about 20% of the total phospholipids and that of PG was 50%. When the isolated membrane was kept at optimal conditions, the CL content increased to 50% and that of PG decreased to 20%. The optimal pH range of the reaction was between 3.0 and 5.0, and the optimal temperature was between 30 and 50℃. The reaction appeared to be completed within three minutes with an apparent Vmax of 74nmoles CL produced/min/mg of membrane protein. The maximum CL content that could be reached in the membrane was no more than 60 % . The reaction was mediated by cardiolipin synthetase in the membrane. The mode of the reaction seemed to be different from that of previous reports (Short S.A. and White D.C. J. Bacteriol. 1972, and Burrit M.F. and Henderson T.O. J. Bacteriol. 1975) in which the reaction was examined by solubilized enzyme with exogenous PG as the substrate. In comparing the mode of reaction in the membrane with that in the solubilized system, it is reasonable to conclude that CL synthetase is buried in the lipid bilayer with its active site facing toward the inside of the cell. The activity of CL synthetase probably is regulated by the pH of the microenvironment brought about by the chemiosmotic potential of the membrane together with the fluidity of the lipid bilayer.