Prebiotic synthesis of phospholipid headgroups. Prebiotic phosphorylation of glycerol delivers glycerol-2,3-cyclic phosphate (G>P) alongside glycerol-3-phosphate (G3P) and glycerol-2-phosphate (G2P). While the hydrolysis of the intrinsically activated G>P has been thoroughly characterized, ring-opening of G>P by nucleophiles other than water remains unexplored. We therefore investigated whether reaction of G>P with Ch in alkaline solution could generate glycerophosphocholines (GPCs). The latter are among the most abundant phospholipid headgroups in eukaryotic membranes, and a potential prebiotic precursor to phosphatidylcholines. Accordingly, an aqueous solution of G>P and Ch at pH 9.5 was heated at 40°C, and the progress of the reaction was followed by 31 P-NMR. Over the course of several weeks, four products were detected: glycerol-3-phosphocholine, G3PC, and glycerol-2-phosphocholine, G2PC, resulting from ring-opening of G>P by Ch (-0.23 ppm and -0.92 ppm, respectively), and G3P and G2P, resulting from hydrolysis of the starting material (4.10 ppm and 3.75 ppm, respectively) (Fig. ). The product distribution varied over time, but the highest cumulative yield of GPCs (0.6%) was obtained after seven weeks (Fig. ). No GPCs were formed when the reaction was carried out at pH 8.5 or 7.5 (Fig. ). In contrast, at pH 10.5 GPCs appeared after just one day (0.7% total yield) (Fig. ). These results show that the reaction between G>P and Ch is subject to specific base catalysis.