1. Diacylglycerol acyltransferase was resolved from rat liver microsomes and separated from glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase. The separation was achieved by sucrose-density-gradient centrifugation of the enzyme preparation which was obtained by molecular-sieve chromatography of microsomes solubilized with a nonionic detergent, Triton X-100. 2. Although diacylglycerol acyltransferase and 1-acylglycerolphosphorylcholine acyltransferase were not separated from each other, the two acyltransferases were distinguishable with respect to heat stability and sensitivity to sulfhydryl-binding reagents. 3. Studies with the diacylglycerol acyltransferase preparation obtained have shown that this enzyme possesses a broad acyl-donor specificity, utilizing saturated, monoenoic, dienoic and tetraenoic fatty acyl-CoA thioesters efficiently. 4. A simplified assay method for diacylglycerol acyltransferase is described.