Oil was extracted from fatty material obtained from Nile perch viscera using the protease Protex 30L. Enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the glyceride fraction was carried out by hydrolysis of extracted oils with lipases from Candida rugosa, Thermomyces lanuginosus and Pseudomonas cepacia. The unusual fatty acid distribution of the oil influenced the apparent lipase specificity to a large extent. In the unhydrolysed oil, only 16% of EPA was in sn-2 position while 51% of palmitic acid was located in this position of the triacylglycerol (TAG) molecules. Non-regioselective lipase from C. rugosa was the most effective in combined enrichment of both EPA and DHA. This was partly because it was able to hydrolyse off palmitic acid from the sn-2 position, which 1-, 3-specific lipases were unable to do. Hydrolysis with C. rugosa lipase enriched EPA from 3 to 6 mol% and DHA from 9 to 23 mol%, with recoveries of 42 and 55%, respectively. The 1-, 3-specific lipase from T. lanuginosus was ineffective in enriching EPA, but gave best DHA enrichment, 38 mol% with a recovery of 39%. DHA was rather equally distributed in sn-1, -2 and -3 positions of TAG. The results show that both the fatty acid specificity and regiospecificity of the lipase as well as the fatty acid distribution of the oil should be considered when choosing the strategy for fatty acid enrichment.