Phosphatidylcholine (PC) was successfully modified by phospholipase A 1 -catalyzed acidolysis with the fatty acids obtained by saponification of fish oil. The resulting phosphatidylcholine contains significant levels of eicosapentaenoic (EPA; C20:5), docosapentaenoic (DPA; C22:5), and docosahexaenoic (DHA; C22:6) acid residues. Modification of the PC was accomplished in a solvent-free system using phospholipase A 1 from Thermomyces lanuginosus/Fusarium oxysporum as the biocatalyst. The effects of variations in the reaction parameters, namely, reaction time, enzyme loading, temperature, and vacuum on the time course of the reaction were investigated. After only 6h of reaction at 55°C, 21.0mol% n−3 polyunsaturated fatty acids (PUFA) (sum of EPA, DPA, and DHA) was incorporated into PC at a loading of the enzyme solution of 10% of the total weight of substrates. As the reaction progressed, incorporation of n−3 PUFA reached a maximum of 28.0mol% at 24h. Reaction times longer than 6h led to higher incorporation of n−3 PUFA but were associated with significant decreases in the yield of PC. Even though application of a vacuum produced a higher yield of PC, there was a parallel decrease in the extent of incorporation of n−3 PUFA into PC.