Diacylglycerol (DAG) acyltransferase catalyses the final and committed step of triacylglycerol biosynthesis. Eukaryotes commonly contain up to three distinct classes of DAG acyltransferases: acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), and phospholipid:diacylglycerol acyltransferase (PDAT). The non-conventional oleaginous yeast, Yarrowia lipolytica, contains at least one homologue of each class and serves as a good model to understand the role of different DAG acyltransferases in the biosynthesis of oil, a critical cellular component that serves as a storage molecule as well as a buffer for free fatty acids. We used gene disruptions in Y. lipolytica and in vitro enzyme assays to confirm the identity of genes encoding all three DAG acyltransferases and demonstrate that together they account for almost all oil biosynthesis and that all three contribute significantly to its oil biosynthesis. In Y. lipolytica ATCC 20362 strain, the total lipid% dry cell weight (DCW) as a percentage of the wild-type strain in pdat, dgat1, dgat2, dgat1/dgat2 double mutant and dgat1/dgat2/pdat triple mutant was 70%, 57%, 36%, 18% and 13%, respectively.This is the first example of DGAT1 contributing significantly to oil biosynthesis in a microorganism. The triple mutant shows significant growth defect in both increased lag phase and slower growth rate, suggesting that oil biosynthesis contributes to normal growth in this strain. Copyright © 2011 John Wiley & Sons, Ltd.