A novel two-step enzymatic method is described in this study to synthesize symmetrical triacylglycerols (TAGs) with arachidonic acid (ARA) at the sn-2 position. The processes included the synthesis of 2-monoacylglycerols (2-MAGs) rich in 2-arachidonoylglycerol (2-AG) by enzymatic ethanolysis and the synthesis of symmetrical TAGs by enzymatic transesterification between 2-MAGs and vinyl palmitate. Under the optimal conditions, desired symmetrical TAGs were obtained at 89% yield. In this study, vinyl palmitate rather than palmitic acid was used as a novel acyl donor to react with 2-MAGs. It was the first study reporting the synthesis of symmetrical TAGs by enzymatic transesterification. The reaction using fatty acid vinyl ester as acyl donor is irreversible and temperature is low. Low-temperature reaction greatly suppressed the acyl migration of 2-MAGs and the irreversible reaction is much more effective compared to reversible reactions using free fatty acid and fatty acid ester as acyl donors. (C) 2015 Elsevier Ltd. All rights reserved.