Cloning and characterization of the Δ9 desaturase (Δ9I) gene of a fungus, Mortierella alpina 1S-4, was previously reported. In this study, two genes encoding Δ9 desaturase homologs were isolated from this fungus. One is a Δ9 desaturase (Δ9II) that exhibits 86% amino acid sequence similarity to Δ9I. Functional analysis involving expression of the encoding gene in Aspergillus oryzae revealed that Δ9II exhibits Δ9 desaturase activity, 18:0 being converted to 18:1Δ9. However, unlike Δ9I, the Δ9II transformant accumulated a low amount of 16:1Δ9. The other homolog is a ω9 desaturase (ω9) that exhibits 56 and 58% amino acid sequence similarity to Δ9I and Δ9II, respectively. On functional analysis with the Aspergillus transformant, it was found that ω9 does not convert 18:0 to 18:1Δ9, but converts 24:0 and 26:0 to 24:1ω9 and 26:1ω9, respectively. On the other hand, Δ9 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 Δ9 desaturase genes were identified. The mutation on the Δ9I 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 Δ9II and ω9 genes. Real-time quantitative PCR (RTQ-PCR) analysis revealed that (1) the transcriptional level of the Δ9I gene in HR222 and T4 was much higher than that in the wild strain until the fifth day of the cultivation periods, (2) the Δ9II gene of the mutants was transcribed until the fourth day at the same level as the Δ9I gene of the wild strain, whereas the Δ9II gene of the wild strain was transcribed at a lower level, and (3) the transcriptional level of the ω9 gene in both the mutants and the wild strain was low, i.e., as low as that of the Δ9II gene of the wild strain. In these Δ9 desaturation-defective mutants, Δ9II is likely to play an important role in Δ9 desaturation.