To utilize n-3 polyunsaturated fatty acid (PUFA) for a wide range of applications, we prepared phospholipids (PLs) containing PUFAs as constituent fatty acids (PUFA-PLs) via commercially available lipase OF-mediated transacylation with PL from soy (Soy-PL) and ethyl ester of PUFA (PUFA-Et). In a preliminary study to evaluate PUFA-incorporation (wt%) on phosphatidylcholine (PC), we observed that dehydration of Soy-PL is critical. PUFA-incorporation in PLs increased with acyl ratio and time. Finally, maximum PUFA-incorporation (47.1 ± 2.1 wt%) was obtained using the following reaction conditions: 2.0 mmol of Soy-PL, a PUFA-Et/Soy-PL acyl ratio of 7, 13 mL of hexane, 2.2 × 10(5) U of lipase OF, 500 rpm of agitation, a temperature of 37°C, and 72 h of reaction time. The analysis of fatty acid composition at the sn-2 position of obtained PL revealed that PUFAs incorporated into Soy-PL localized to the sn-2 position of the PL molecule in spite of using lipase OF whose positional specificity is random for triacylglycerol.