Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3omega9) was attained with a mutant fungus, Mortierella alpina JT-180, derived from Delta12 desaturation activity-defective and Delta6 desaturation activity-enhanced M. alpina M209-7. Production of 20:3omega9 by JT-180 was 1.4 times greater than that of the parent strain M209-7. This is thought to be due to its enhanced Delta5 desaturation activity, which was 3.3 times higher than that of M209-7. In both strains, 78.5-80.4% of the total lipids comprised triacylglycerol (TG), and 76.6-79.0% of 20:3omega9 was present in TG. Comparing the fatty acid compositions among various lipid species, the highest percentages (24.1-37.6%) of 20:3omega9 in total lipids were found in phosphatidylcholine. For optimization of 20:3omega9 production by JT-180, a glucose concentration of 4% in the culture medium and shifting of the growth temperature from 28degreesC to 20degreesC on the 2nd day were shown to be effective. Under optimal conditions, 20:3omega9 production by JT-180 reached 1.92 g/l culture medium in a 10-1 jar fermentor (corresponding to 81.5 mg/g dry mycelia and 18.3% of total fatty acids), which is greater than that reported previously from M209-7 (1.65 g/l).