CTP:phosphocholine cytidylyltransferase (CCT) is an enzyme critical for cellular phosphatidylcholine (PC) synthesis, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline. We have isolated a cDNA encoding an isoform of CCT from Drosophila melanogaster and expressed the recombinant native and 6 x -His-tagged forms using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Immunoblot using anti-phospho amino acid antibodies reveals the enzyme is phosphorylated on serine and threonine residues, but not tyrosine. The purified native enzyme exhibits a V(max) value of 1352+/-159 nmol CDP-choline/min/mg, a K(m) value of 0.50+/-0.09 mM for phosphocholine, and a K' (Hill constant) value of 0.72+/-0.10 mM for CTP. The 6 x -His-tagged enzyme has similar properties with a V(max) value of 2254+/-253 nmol CDP-choline/min/mg, a K(m) value of 0.63+/-0.13 mM for phosphocholine and a K' for CTP equal to 0.81+/-0.20 mM. Each form of the enzyme was activated to a similar extent by synthetic PC vesicles containing 50 mol% oleate. The efficiency of lipid activation was greatest using PC vesicles containing diphosphatidylglycerol (DPG), significantly less efficient activation was seen when phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators.