Mono- (MAG) and diacylglycerols (DAG) are of nutritional interest. MAG and DAG containing eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were produced in a solvent-free system via glycerolysis of menhaden oil catalyzed by Novozym 435. The effect of the molar ratio of glycerol to oil, enzyme concentration, and reaction temperature on MAG and DAG production was assessed. The optimal temperature was in the range of 55-70 _C for production of both acylglycerols. The increase in the substrates molar ratio led to a decrease in MAG and DAG content. The enzyme concentration was fixed at the lowest level evaluated (5%, by weight of substrates). High content of MAG (25% by weight) and DAG (41% by weight) containing, respectively, 12.46% EPA and 11.16% DHA, and 14.57% EPA and 13.70% DHA, were produced after 24 h at 70 _C, with 5% of lipase (by weight of substrate) and a glycerol-to-oil molar ratio of 1:1. For this reaction, a molar triacylglycerol (TAG) conversion of about 60% was achieved at equilibrium (10 h).