The gamma-linolenic acid-producing fungus Mortierella isabellina 6-22 is an important industrial strain. To clarify the biosynthetic pathways for polyunsaturated fatty acids in this strain, a disruption vector pD4MI6, including 5' and 3' regions of the fatty acid Delta(6)-desaturase open reading frame as homologous recombination elements and the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph) as selectable marker, was successfully constructed. Following transformation of pD4MI6 into the hygromycin B-sensitive recipient strain M. isabellina 6-22-4, a Delta(6)-desaturase gene-defective mutant strain was selected that was unable to produce gamma-linolenic acid as determined by gas chromatography and molecular analysis. The morphology and physiology of the mutant, such as colony shape, color, and growth rate, were changed dramatically compared with that of strain M. isabellina 6-22-4.