In vitro characterizations of rat liver microsomal acyl-CoA:sn-glycerol 3-phosphate acyltransferase (EC 22.214.171.124) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 126.96.36.199) (DHAP acyltransferase) activities were performed in order to test the hypothesis that these two activities in liver microsomes may be dual functions of a single enzyme and to estimate the relative contributions of the glycerol-P and DHAP pathways for hepatic microsomal glycerolipid synthesis. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 0.14 mm for glycerol-P with a V of 2.8 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 0.3 mm for DHAP with a V of 0.62 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 0.12 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki = 1.4 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities showed great similarities in their pH dependence, acyl-CoA chain length and concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Taken as a whole the data strongly suggest that liver microsomal glycerol-P and DHAP acyltransferase activities actually represent dual catalytic functions of a single microsomal enzyme. Similar kinetic and inhibition studies indicate that this finding is also valid for the two acyltransferase activities from intestinal mucosa, lung, kidney, and brain. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in liver suggest that the in vivo ratio of glycerol-P to DHAP acylation in liver microsomes should be greater than 84:1. © 1977.