The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.