Two lipases (Lip A and Lip B), were purified from a commercial lipase preparation produced by Candida rugosa and partially characterized. The purified lipases were immobilized on Duolite A 568 and used in the selective esterification of cholesterol with free fatty acids from sardine fish oil. The results showed that Lip A and Lip B preferentially esterified saturated and monounsaturated fatty acids allowing a 3.4-fold (Lip B, 24 h) and 4-fold (Lip A, 10 h) enrichment of docosahexaenoic acid in the remaining free fatty acid fraction. Selectivity towards cicosapentaenoic acid was less pronounced. By this selective esterification docosahexaenoic acid was concentrated from 7.4 to 32% with a recovery of 95% of its initial content in sardine fish oil.