This paper studies the synthesis of 2-monoacylglycerols (2-MAG), rich in polyunsaturated fatty acids (PUFAs) by alcoholysis of fish oils with ethanol, catalyzed by 1,3 specific lipases. Cod liver and tuna oils were used as PUFA rich substrates, and the influence of the following variables was studied: (i) ethanol purity (commercial, 96% (v/v); absolute, 0.5% water, and absolute dry, 0.02% water), (ii) the lipase used (Novozym 435 from Candida antarctica; lipase D from Rhizopus oryzae and lipase EU 093 or Rd, from Rhizopus delemar), (iii) ethanol/oil molar ratio, (iv) treatment intensity (TI = lipase amount x reaction time/oil amount), and (v) solvent/oil ratio. High yields in 2-MAG (90.3%) were obtained with 96% ethanol and lipase Novozym 435, which behaves as 1,3 specific when a great excess of ethanol is used. Nevertheless, the water accompanying ethanol produces free fatty acids (FFA), which make the posterior separation of 2-MAG difficult. 2-MAG yield of 75% was reached with lipases D and Rd immobilized on Accurel MP-1000, using absolute ethanol; in these conditions free fatty acid formation and acyl-migration were not observed. This yield was obtained by using an ethanol/oil molar ratio of 11 and 6 mL of acetone per gram of oil; the alcoholysis rate reached a minimum value when no solvent (acetone) was used; nevertheless, the 2-MAG yield at equilibrium did not seem to be influenced by the acetone/oil ratio. The separation of 2-MAG from ethyl esters was carried out by silica gel chromatography and solvent extraction. The first method obtained 2-MAG with 96% purity and 85% yield while smaller values were obtained by the second method (89% purity and 77% yield). Nevertheless, much higher amounts of 2-MAG can be obtained by the latter using less volume of solvents. Finally, the positional analysis of the two initial oils revealed that all the PUFAs that were initially located in position 2 of triacylglycerols stay in this position in the final 2-MAG. This revealed that lipases D and Rd are completely 1,3 specific and that no acyl-migration occurred. (C) 2008 Elsevier Ltd. All rights reserved.