A triacylglycerol synthetase complex made up of acyl-CoA synthetase, acyl-CoA:monoacylglycerol acyltransferase, and acyl-CoA:diacylglycerol acyltransferase has been solubilized by sodium taurocholate and isolated by chromatography on phenyl-Sepharose. For this purpose microsomes of the villus cells of rat intestinal mucosa were dissolved in 2% sodium taurocholate prepared in 1 M (NH4)2SO4 and 25 mM Tris-HCl (pH 8.5) (buffer A). After dialysis against buffer A, the sample was loaded on a phenyl-Sepharose column and the enzyme complex was eluted with 25 mM Tris-HCl (pH 8.5) (buffer B). The enzymatically active fractions were pooled and rechromatographed on a Bio-Gel A-0.5m column equilibrated with buffer B and 150 mM NaCl. The recovered triacylglycerol synthetase complex accounted for over 75% of the original enzyme activity and represented a 145-fold purification from villus cells and a 10-fold purification from microsomes. It exhibited maximal activity at pH 8.0-9.0. On the basis of Bio-Gel A-0.5m chromatography the three enzymic activities appeared as a single fraction in the molecular weight range of 350 000-375 000. The complex migrated as a single peak on high performance liquid chromatography on an ion-exchange column using a NaCl gradient. The ratio of the activities of the three enzymes remained constant during the purification. The purified enzyme complex lost about 50% of its diacylglycerol acyltransferase activity on storage for 2 weeks at -20 degrees C in presence of phenylmethylsulfonyl fluoride.