In cyanobacteria, the biosynthesis of unsaturated fatty acids is initiated by delta 9 acyl-lipid desaturase which introduces the first double bond at the delta 9 position of a saturated fatty acid that has been esterified to a glycerolipid. We have cloned genes, designated desC, for delta 9 acyl-lipid desaturases from two cyanobacteria, namely Anabaena variabilis and Synechocystis sp. PCC 6803. These desaturases, when expressed in Escherichia coli, desaturated stearic acid to yield oleic acid at the C-1 positions of phosphatidylethanolamine and phosphatidylglycerol, but did not desaturate palmitic acid, palmitoleic acid, and cis-vaccenic acid. These results indicate that the delta 9 acyl-lipid desaturases are specific to stearic acid esterified at the C-1 position of a glycerolipid and are nonspecific with respect to the polar head group of the glycerolipid. The deduced amino acid sequences of the delta 9 acyl-lipid desaturases are similar in part to those of stearoyl-CoA desaturases of the rat, the mouse, and Saccharomyces cerevisiae, but not to those of acyl-(acyl-carrier-protein) desaturases of higher plants.