Cloning and characterization of the Delta 9 desaturase (Delta 9I) gene of a fungus, Mortierella alpina 1S-4, was previously reported. In this study, two genes encoding Delta 9 desaturase homologs were isolated from this fungus. One is a Delta 9 desaturase (Delta 9II) that exhibits 86% amino acid sequence similarity to Delta 9I. Functional analysis involving expression of the encoding gene in Aspergillus oryzae revealed that Delta 9II exhibits Delta 9 desaturase activity, 18:0 being converted to 18:1 Delta 9. However, unlike Delta 9I, the Delta 9II transformant accumulated a low amount of 16:1 Delta 9. The other homolog is a omega 9 desaturase (omega 9) that exhibits 56 and 58% amino acid sequence similarity to Delta 9I and Delta 9II, respectively. On functional analysis with the Aspergillus transformant, it was found that omega 9 does not convert 18:0 to 18:1 Delta 9, but converts 24:0 and 26:0 to 24:1 omega 9 and 26:1 omega 9, respectively. On the other hand, Delta 9 desaturation-defective mutants characterized by accumulation of 18:0 were derived from M. alpina 1S-4 with a chemical mutagen, and the mutated sites of the Delta 9 desaturase genes were identified. The mutation on the Delta 9I gene was assumed to cause an amino acid replacement (W136Stop, G265D, and W360Stop) in the mutants (HR222, T4, and ST56), respectively. In these mutants, there was no mutated site on the Delta 9II and omega 9 genes. Real-time quantitative PCR (RTQ-PCR) analysis revealed that (1) the transcriptional level of the Delta 9I gene in HR222 and T4 was much higher than that in the wild strain until the fifth day of the cultivation periods, (2) the Delta 9II gene of the mutants was transcribed until the fourth day at the same level as the Delta 9I gene of the wild strain, whereas the Delta 9II gene of the wild strain was transcribed at a lower level, and (3) the transcriptional level of the omega 9 gene in both the mutants and the wild strain was low, i.e., as low as that of the Delta 9II gene of the wild strain. In these Delta 9 desaturation-defective mutants, Delta 9II is likely to play an important role in Delta 9 desaturation.