The properties of rat liver microsomal CDPethanolamine: 1,2-diacylglycerol ethanolamine-phosphotransferase (ethanolaminephosphotransferase; EC 2.7.8.1) are studied with respect to metal ion and substrate concentration. The enzyme requires magnesium (20 mM) or manganese (1 mM) ions for optimum activity. Manganese ions are better activators of ethanolaminephosphotransferase activity than are magnesium ions. Calcium 1 mM) inhibits the magnesium-activated ethanolaminephosphotransferase by 86% and the manganese-activated enzyme by 57%. The Km for CDPethanolamine is 2.4 and 0.7 x 10(-4) M, in the presence of magnesium or manganese ions, respectively. ATP plus pantetheine significantly inhibit the manganese-activated ethanolaminephosphotransferase, while the magnesium-activated enzyme is inhibited by ATP plus pantetheine and slightly stimulated by ATP plus CoA. In the presence of either metal ion, ATP itself inhibits enzyme activity, while CoA or pantetheine when added alone have no effect. Evidence is presented indicating that the inhibition of ethanolaminephosphotransferase activity by ATP plus CoA or ATP plus pantetheine is not due to the formation of acyl-CoA or acyl-S-pantetheine esters. At the present time, however, the true mechanism of inhibition is unknown. The results indicate that the cellular levels of ATP, CoA, pantetheine, magnesium, manganese, and calcium ions may all play a role in the regulation of phosphatidylethanolamine biosynthesis.