A third gene (Delta9-3) encoding a fatty acid Delta9-desaturase was isolated from the oil-producing fungus Mortierella alpina. The predicted protein of 512 aa shared 53% sequence identity with the two fatty acid Delta9-desaturases, ole1p and ole2p, already described in this organism and contained three histidine boxes, four putative transmembrane domains and a C-terminal cytochrome b(5) fusion that are typical of most fungal membrane-bound fatty acid desaturases. However, unlike the M. alpina ole1 and ole2 genes, the Delta9-3 ORF failed to complement the Saccharomyces cerevisiae ole1 mutation. GC-MS analysis of fatty-acid-supplemented ole1 yeast transformants containing the Delta9-3 gene indicated that this enzyme had negligible activity with endogenous palmitic acid (16:0) as substrate and moderate activity (30-65% desaturation) with endogenous stearic acid (18:0). Yeast transformants overexpressing any one of the three M. alpina fatty acid Delta9-desaturase genes or the S. cerevisiae OLE1 gene produced low amounts of hexacosenoic acid [26:1(n-9)], a fatty acid that is not normally present in yeast cells. It follows that these Delta9-desaturases may also display low n-9 desaturation activity with very long-chain saturated fatty acid substrates. Conversely, high levels of desaturase in the endoplasmic reticulum membrane of these yeast transformants may increase the availability of suitable monounsaturated substrates for fatty acid elongation.