This review summarizes the characteristics of two subclasses of phosphatidylserine synthases: subclass I of gram-negative bacteria and subclass II of gram-positive bacteria. Unlike other phospholipid biosynthetic enzymes, the phosphatidylserine synthases of gram-negative bacteria, the enzyme from Escherichia coli has been extensively examined and characterized, are associated with the ribosomal fraction of cell lysates. Enzymes from gram-positive bacteria are membrane-bound, and the structural gene of membrane-bound synthase of Bacillus subtilis has been cloned and used in our laboratory for replacement with the E. coli counterpart. This review discusses the possible regulatory mechanisms of phosphatidylethanolamine synthesis in E. coli, which are closely related to the subcellular localization and properties of phosphatidylserine synthase, and highlights the cross-feedback regulatory model which assumes two forms of phosphatidylserine synthase (only molecules bound with acidic phospholipids of the membrane are active in phosphatidylserine synthesis, whereas others in the cytoplasm are latent). In addition, considerations of the origin and evolution of the two vastly different subclasses of phosphatidylserine synthases of bacteria are also presented.