Triacylglycerols are the most important storage lipids in most plants and animals. Acyl-CoA:diacylglycerol acyltransferase (EC 220.127.116.11) catalyzes the final step of the pathway of triacylglycerol synthesis and is the only step which is unique to this process. Diacylglycerol acyltransferase is required for the synthesis of storage oil in a wide range of oil-bearing seeds and fruits and in floral structures such as petals, anthers and pollen. We describe the first cloning and functional expression of a cDNA encoding diacylglycerol acyltransferase from a plant. The cDNA, cloned from Arabidopsis thaliana, encodes a 520 amino acid protein with a predicted molecular mass of 59.0 kDa which shares 38% amino acid sequence identity with diacylglycerol acyltransferase from mouse. When expressed in insect cell cultures, the protein catalyzes the synthesis of [14C]triacylglycerol from [14C]diacylglycerol and acyl-CoA. Primer extension analysis revealed that the transcription begins 225 bases before the translation start site, yielding an unusually long 5' untranslated region. The gene is expressed in a wide range of tissues but most strongly in developing embryos and petals of flowers.