The mono trans geometrical isomer of eicosapentaenoic acid, 5c,8c,11c,14c,17t-eicosapenntaenoic acid (20:5Delta5c,8c,11c,14c,17t), was synthesized by fatty acid microbial conversion using a Delta12-desaturase defective mutant of an arachidonic acid (AA)-producing fungus, Mortierella alpina 1S-4. The substrate for the bioconversion, a geometrical isomer of linolenic acid, was prepared by isomerization of linseed oil methyl ester by the nitrous acid method, followed by purification on a AgNO3-Silica gel column. The structure and double bond geometry were identified after hydrazine reduction followed by permanganate oxidation to 20:5Delta5c, 8c,11c,14c,17t. The biosynthetic route from 18:3Delta6c, 9c,12t to 20:5Delta5c,8c,11c,14c,17t was presumed to mimic the route from linoleic acid to arachidonic acid.