Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K(m) and IC(50) values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [(32)P]H(3)PO(4) demonstrated that Ser(568) and Ser(571) were two major phosphorylation sites, and additional studies demonstrated that Ser(568) was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser(568) (S568A) but not Ser(571) significantly increased hCTPS2 activity, demonstrating that Ser(568) is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V(max) of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser(568) as a major site of CTPS2 regulation by phosphorylation.