The activities of the proposed triacylglycerol synthetase complex, acyl-CoA ligase, acyl-CoA acyltransferase (AAT), monoacylglycerol acyltransferase (MGAT), and diacylglycerol acyltransferase (DGAT), coeluted upon Cibacron blue 3GA-agarose affinity chromatography of detergent-solubilized rat intestinal microsomes. The AAT activity is associated with a 54-kDa protein, that binds covalently an acyl group from acyl-CoA via a thiol ester linkage (Lehner, R. and Kuksis, A. (1993) J. Biol. Chem. 268, 24726-24733). Reagents that prevent the acyl-AAT formation inhibit triacylglycerol synthesis as does the removal of AAT from the complex by immunoprecipitation. In the absence of mono- and diacylglycerol acceptors, the acyl group is transferred to water. It is proposed that triacylglycerol synthesis proceeds via a sequential transfer of acyl groups from acyl-CoA ligase to the AAT, from which they are passed to the mono- and diacylglycerol acyltransferases for incorporation into the di- and triacylglycerols depending on the availability of the acyl acceptors.