Two kinds of lipases, AK-lipase and HU-lipase, produced by two different Pseudomonas fluorescens strains, AK102 and HU380, respectively, were evaluated as to fatty acid hydrolysis specificity using six types of oil containing higher amounts of C20 fatty acids such as arachidonic acid (5,8,11,14-eicosatetraenoic acid, AA, or 20:4 omega 6), dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid, DGLA, or 20:3 omega 6), 5,8,11,14,17-eicosapentaenoic acid (EPA or 20:5(0), mead acid (5,8,11-eicosatrienoic acid, MA, or 20:3 omega 9), 8,11-eicosadienoic acid (20:2 omega 9) and 8,11,14,17-eicosatetraenoic acid (20:40). Although HU-lipase did not show any specificity for C20 fatty acids with respect to the presence or absence of a Delta 5 unsaturated bond, it exhibited comparatively low reactivity for 4,7,10,13,16,19-docosahexaenoic acid (DHA or 22:6 omega 3). In contrast, AK-lipase was less reactive for C20 fatty acids with a Delta 5 unsaturated bond. However, the specificity of hydrolysis of AK-lipase gradually decreased as the reaction proceeded. Utilizing this fatty acid specificity, we concentrated either EPA or DHA from fish oils containing both EPA and DHA by means of lipase-catalyzed hydrolysis and urea adduction. Hydrolysis and urea adduction of refined cod oil including 12.2% EPA and 6.9% DHA with HU-lipase provided free fatty acids with 43.1% EPA and 7% DHA, respectively. The resulting yield of concentrated total fatty acids comprised 2.6% of the fatty acids from the cod oil. Thus, EPA was particularly concentrated in the fatty acids derived from refined cod oil on partial hydrolysis with HU-lipase followed by urea adduction. On the other hand, hydrolysis of cuttlefish oil with AK-lipase followed by urea adduction increase slightly the EPA composition from 14.2% to 16.8%, and markedly enhanced the composition of DHA from 16.3% to 44.6% in the hydrolyzed fatty acids. The yield of purified total fatty acids by urea concentrate was 9.4% of the fatty acids from the cuttlefish oil. Thus, DHA was particularly concentrated in the fatty acids derived from on partial hydrolysis with AK-lipase followed by urea adduction. We concluded that EPA and DHA concentrates can be easily and inexpensively obtained using HU-lipase and AK-lipase, respectively. Furthermore, it might be possible to separate and concentrate C20 polyunsaturated fatty acids (PUFAs) with or without a Delta 5 double bond from PUFAs rich oils including both fatty acids.