In oleaginous fungus Cunninghamella echinulata, Δ9-fatty acid desaturase introduces the first double bond into a saturated fatty acid. Three distinct genes, designated as d9dma, d9dmb and d9dmc, all encoding putative Δ9-fatty acid desaturases were isolated from this strain. The predicted proteins showed 79-87 % identity to other fungal Δ9-fatty acid desaturases. They all contain three conserved histidine boxes, C-terminal cytochrome b 5 fusion and four transmembrane domains characteristic of Δ9-desaturase. Each putative Δ9-desaturase gene from C. echinulata was able to complement the ole1 mutation in Saccharomyces cerevisiae L8-14C through heterologous expression. Analysis of the fatty acid composition of the transgenic yeast revealed that the conversion rates of 16:0 and 18:0 by D9DMA were obviously higher than those of D9DMB and D9DMC. In addition, D9DMA, D9DMB and D9DMC all had a substrate preference for 18:0 compared with 16:0. Of interest, D9DMA could saturate 12:0, 14:0, 16:0, 17:0, 18:0 and 20:0, while D9DMB saturated 14:0, 16:0, 17:0, 18:0 and 20:0. We also noticed that the transcriptional level of d9dma in C. echinulata was stimulated by cell growth but not by decline in temperature. In contrast, expression of d9dmb and d9dmc was regulated by neither cell growth nor decline in temperature in this strain.