Triacylglycerols (TAGs) are abundant storage compounds found in seeds from oleaginous plants. Their accumulation strongly depends on the activity of acyl-CoA:diacylglycerol acyltransferases (DGAT), and of phospholipid:diacylglycerol acyltransferases (PDAT). Three families of DGATs are known to date. DGAT1 and 2 are membrane proteins, and their localization and contribution to specific fatty acid accumulation in the seed oil has been described in several plants. DGAT3 represents a third evolutionary independent family of enzymes only found in the plant kingdom. They possess DGAT activity (1), and are apparently involved in recycling of 18:2 and 18:3 FAs into TAGs (2). Contrary to DGAT1 and 2, DGAT3 lack transmembrane regions and their cellular and subcellular localization remains unknown. DGAT3 possesses a putative chloroplast transit peptide, and a thioredoxin-like ferredoxin domain typical for a class of ferredoxins harboring a [2Fe-2S] cluster. The recombinant DGAT3 from Arabidopsis thaliana (AtDGAT3; At1g48300) produced from Escherichia coli, although very unstable, exhibits DGAT activity in vitro, using diacylglycerol as acceptor and acyl-CoA as donor. A shorter protein version Δ46AtDGAT3 was more stable in vitro, allowing biochemical and spectroscopic characterization. Our results demonstrate the presence of a [2Fe-2S] cluster in the protein. To date, AtDGAT3 is the first metalloprotein described as a DGAT (3). Using specific antibodies, we have found that AtDGAT3 is expressed in young seedlings (3) and we have determined the subcellular localization of the enzyme using electron microscopy employing immunogold labelling. To further elucidate the physiological function, we also have characterized the lipids from A. thaliana lines in which AtDGAT3 has been knocked out.