The activity of phosphatidylserine synthase from Escherichia coli depends significantly on the nature and level of the lipids in the matrix, at which the enzyme is operating. To elucidate the role of anionic lipids in the regulation of PtdSer synthase, its activity was studied in mixed micelles containing phosphatidylglycerol (one charge) or diphosphatidylglycerol (two charges), the two main anionic membrane lipids in E. coli. Membrane association and activity of PtdSer synthase were increased by the two lipids, indicating their essential role in the positive regulation mechanism of the phosphatidylethanolamine level in the E. coli membrane.