1-Acylglycerolphosphate acyltransferase (Ec 2.3.1-) and 1-acylglycerolphosphorylcholine acyltransferase (EC 184.108.40.206) of rat liver microsomes were separated from each other. The separation was achieved by sucrose density gradient centrifugation of the enzyme preparation that was obtained by solubilizing microsomes with a nonionic detergent, Triton X-100, and subjecting the solubilized microsomes to molecular-sieve chromatography. The two acyltransferases are distinguishable from each other also with respect to their stabilities to heat and to Triton X-100. Hence, it is concluded that these acyltransferases are distinct enzymes. These results, together with our previous finding that glycerolphosphate acyltransferase is also a separate enzyme, demonstrate the presence of distinct acyltransferases responsible for the acylation of the different acyl acceptors. Furthermore, the acyl-donor specificities of these acyltransferases provide the enzymatic basis for the nonrandom distribution of fatty acids in naturally occurring glycerolipids.