The acyl CoA:sn glycerol 3 phosphate acyltransferase (EC 126.96.36.199) (glycerol P acyltransferase) and acyl CoA:dihydroxyacetone phosphate acyltransferase (EC 188.8.131.52) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol P acyltransferase activity showed an apparent K(m) of 8 μM for glycerol P with a V(max) of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent K(m) of 40 μm for DHAP with a V(max) of 9.7 nmol/min/mg. Glycerol P was a competitive inhibitor (K(i) = 7.2 μM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (K(i) = 92 μM) of the glycerol P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl CoA chain length dependence, thermolability, and inactivation by N ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol P to DHAP acylation should be greater than 24:1.