Technical Abstract: 1,2-Diacylglycerols (DAG) are the native substrates for the diacylglycerol acyltransferase. While DAG containing saturated or unsaturated fatty acids are relatively easy to make chemically, it is difficult to chemically synthesize DAG containing hydroxy fatty acids in specific positions on the glycerol backbone. An alternate approach is to start from acylglycerols containing hydroxy fatty acids, and selectively remove fatty acid chains to obtain the desired acylglycerol. The purpose of this study was to identify optimal parameters for lipase-catalyzed methanolysis of triricinolein to produce 1,2(2,3)-diricinolein, the substrate for biosynthesis of triricinolein in castor (Ricinus communis). We tested four different immobilized lipases, using n-hexane and diisopropyl ether (DIPE) as reaction media, and three different water activities. We followed the consumption of triricinolein and the formation of diricinolein, methyl ricinoleate and ricinoleic acid during the course of the reaction. Penicillium roquefortii lipase gave the highest yield of 1,2(2,3)-diricinolein and this lipase showed the highest specificity for the studied reaction, i.e. high selectivity for reaction with triricinolein, but low for diricinolein. The diacylglycerol produced can be acylated by the diacylglycerol acyltransferase from Arabidopsis thaliana. Therefore, the product of the lipase reaction is a suitable substrate for acyltransferase reactions.