The Arabidopsis ADS gene family contains nine genes encoding fatty acid desaturase-like proteins. The biological function of only one member of the family, AtADS3/FAD5 (At3g15850) is known and this gene encodes the plastidic palmitoyl-monogalactosyldiacylglycerol Δ7-desaturase. We cloned seven members of the gene family that are predicted not to have a chloroplast transit peptide and expressed them in the yeast Sacchromyces cerevisiae. All seven have previously undescribed desaturase activity on very long chain fatty acid (VLCFA) substrates and exhibit diverse regiospecificity, catalyzing introduction of double bonds at n-6 (AtADS4, At1g06350), n-7 (AtADS1.3, At1g06100 and AtADS4.2, At1g06360), n-9 (AtADS1, At1g06080 and AtADS2, At2g31360) or Δ9 positions (AtADS1.2, At1g06090 and AtADS1.4, At1g06120). Through forward and reverse genetics it was shown that AtADS2 is involved in the synthesis of the 24:1n-9 and 26:1n-9 components of seed lipids, sphingolipids and the membrane phospholipids phosphatidylserine (PS), and phosphatidylethanolamine (PE). Plants deficient in AtADS2 expression showed no obvious phenotype when grown under normal growing conditions, but showed an almost complete loss of PE(42:4), PS(42:4), HexCer(42-2)-2, HexCer (42:2)-3 and GIPC(42:2)-3, lipid species that contain the VLCFA 24:1n-9, and GIPC(44:2)-3, a lipid containing 26:1n-9. Plants deficient in AtADS1 expression had reduced levels of 26:1n-9 in seed lipids, but no significant changes in leaf phospholipids or sphingolipids were observed. These findings indicate that the C24 and C26 monounsaturated VLCFAs of Arabidopsis result primarily from VLCFA desaturation rather than elongation of long chain (C≤18) monounsaturated fatty acids.