The catalytic potential of phospholipase A(2) (PLA(2)) for the synthesis of phospholipids with defined fatty acid structure in the sn-2 position has been underestimated hitherto because of very low conversion in most organic solvents. One of the most suitable solvents for PLA(2)-catalyzed phospholipid synthesis is glycerol. With the aim to analyze the effect of several interacting reaction parameters on the product yield, we studied the conversion of 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) with oleic acid as model reaction in mixtures of glycerol and methanol or ethanol by methods of experimental design. PLA(2) from porcine pancreas (ppPLA(2)) and from bee venom (bvPLA(2)) were compared as catalysts. For each of the four systems, nine variables were evaluated using Plackett-Burman designs. The most significant four variables were used for subsequent modified D-optimal designs with 30 runs, yielding regression equations for describing the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine as a function of the variables. In both solvent systems ppPLA(2) was more appropriate for the acylation reaction than bvPLA(2). Methanol proved to be more convenient as co-solvent than ethanol. The catalysis by ppPLA(2) was more sensitive toward the variables temperature and concentration of Tris-HCl, whereas the reaction time and enzyme activity were more important in the acylation by bvPLA(2). Conversion up to 87 (ppPLA(2)) and 50% (bvPLA(2)) can be anticipated. (C) 2014 Elsevier Inc. All rights reserved.