Biosynthesis of polyunsaturated fatty acids in C. elegans is initiated by the introduction of a double bond at the Delta 9 position of a saturated fatty acid. We identified three C. elegans fatty acid desaturase genes related to the yeast Delta 9 desaturase OLE1 and the rat stearoyl-CoA desaturase SCD1. Heterologous expression of all three genes rescues the fatty acid auxotrophy of the yeast Delta 9 desaturase mutant ole1. Examination of the fatty acid composition of the transgenic yeast reveals striking differences in the substrate specificities of these desaturases. Two desaturases, FAT-6 and FAT-7, readily desaturate stearic acid (18:0) and show less activity on palmitic acid (16:0). In contrast, the other desaturase, FAT-B, readily desaturates palmitic acid (16:0), but shows nearly undetectable activity on the common Delta 9 substrate stearic acid. This is the first report of a palmitoyl-CoA-specific membrane fatty acid desaturase.