Three different cDNA sequences, designated OepFAD2-3, OepFAD2-4 and OepFAD2-5, encoding three microsomal oleate desaturases (FAD2) have been isolated from olive (Olea europaea cv. Picual). Sequence analysis and functional expression in yeast of the corresponding cDNAs confirm that they encode microsomal oleate desaturases. Gene expression and lipid analysis indicate that these three genes are not involved in the linoleic acid present in seed lipids, while OeFAD2-5, together with OeFAD2-2, contributes mostly to the linoleic acid present in the mesocarp and, therefore, in the olive oil. Our results have also shown that olive FAD2-3, FAD2-4 and FAD2-5 gene expression is not only spatially and temporally regulated in olive fruit, but also is cultivar-dependent, as well as regulated by water regime, temperature, light and wounding. All these data suggest specialized physiological roles for the olive FAD2 gene family members with respect to both aspects of the biosynthesis of the linoleic acid, either present in storage lipids that constitute the olive oil or being part of membrane lipids, which are involved in the response to abiotic stresses, and highlight the differences on FAD2 gene regulation between oilseeds and oil fruits.