The synthesis of CDP-diacylglycerol by CTP: phosphatidate cytidylyltransferase (EC 126.96.36.199) has been studied in microsomes isolated from hog mesenteric lymph node lymphocytes. The properties of this enzyme were found to be similar in many respects to that described in rat liver microsomes; however, it is not stimulated by GTP or any other nucleotides under normal assay conditions. The enzyme requires that the phosphatidate be emulsified in a cationic detergent for optimal activity. The quaternary ammonium phospholipids lecithin and sphingomyelin were found to stimulate the formation of CDP-diacylglycerol even in the presence of optimal cationic detergent. Other phospholipids or detergents had no effect or were inhibitory to the reaction. Only in the presence of either lecithin or sphingomyelin did nucleotides such as ATP, GTP, UTP, and ITP stimulate the formation of CDP-diacylglycerol. The Km and Vmax for CTP were found to be 0.6 and 1.2 mM, respectively, while the apparent Km and Vmax for phosphatidate were 0.65 and 1.2 mM. Magnesium was found to be the only metal ion that stimulated the reaction, with an optimal concentration of 20 mM. Fluoride ions at 20 mM inhibited the reaction to the extent of 70%. The enzyme was found to be very unstable when the isolated microsomes were stored at -20 degrees C for 24 h, losing approximately 75% of its activity.