A series of new phospholipids with polar head groups have been synthesized by enzymatic transphosphatidylation of 1,2-dioleoyl-sn-glycerophosphocholine and identified by 1H NMR and MALDI-TOF-MS. The acceptor alcohols were N- or C2-substituted derivatives of ethanolamine (diethanolamine, triethanolamine, serinol, Tris, BisTris). Phospholipases D from cabbage (PLDcab) and Streptomyces sp. (PLDStr) were compared with respect to product yield and purity as well as the initial rates in transphosphatidylation and competing hydrolysis. In all reactions, PLDStr showed a remarkably higher transphosphatidylation activity than PLDcab. However, higher yields of the phospholipids with diethanolamine, triethanolamine, and serinol were obtained by PLDcab because PLDStr resulted in the additional formation of diphosphatidyl derivatives. In the synthesis of the Tris and BisTris derivatives, PLD(Str) was much more appropriate because voluminous head group alcohols (>129A3) are poorly converted by PLDcab. With BisTris as acceptor alcohol two regioisomeric forms of phosphatidyl-BisTris were obtained.