Arbuscular mycorrhizal (AM) fungi are oleaginous organisms, and the most abundant fatty acyl moiety usually found in their lipids is palmitvaccenic acid (16:1Δ11cis). However, it is not known how this uncommon fatty acid species is made. Here, we have cloned two homologs of Lepidopteran fatty acyl-CoenzymeA Δ11 desaturases from the AM fungus Rhizophagus irregularis. Both enzymes, DES1 and DES2, are expressed in intraradicle mycelium and can complement the unsaturated fatty acid-requiring auxotrophic growth phenotype of the Saccharomyces cerevisiae ole1Δ mutant. DES1 expression leads almost exclusively to oleic acid (18:1Δ9cis) production, whereas DES2 expression results in the production of 16:1Δ11cis and vaccenic acid (18:1Δ11cis). DES2 therefore encodes a Δ11 desaturase that is likely to be responsible for the synthesis of 16:1Δ11cis in R. irregularis