The synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts with the formation of carbamoyl phosphate from glutamine and CO. Next, aspartate carbamoyltransferase catalyzes a condensation reaction between aspartate and carbamoyl phosphate to form carbamoyl aspartic acid, which is cyclized into 4,5-dihydroorotic acid by dihydroorotase. The latter is converted to orotate by dihydroorotate oxidase. The net reaction is:
:(S)-Dihydroorotate + O → Orotate + HO
Orotate is covalently linked with a phosphorylated ribosyl unit. The covalent linkage between the ribose and pyrimidine occurs at position C of the ribose unit, which contains a pyrophosphate, and N of the pyrimidine ring. Orotate phosphoribosyltransferase (PRPP transferase) catalyzes the net reaction yielding orotidine monophosphate (OMP):
:Orotate + 5-Phospho-α-D-ribose 1-diphosphate (PRPP)  →  Orotidine 5'-phosphate + Pyrophosphate
Orotidine 5-monophosphate is decarboxylated by orotidine-5-phosphate decarboxylase to form uridine monophosphate (UMP). PRPP transferase catalyzes both the ribosylation and decarboxylation reactions, forming UMP from orotic acid in the presence of PRPP. It is from UMP that other pyrimidine nucleotides are derived.  UMP is phosphorylated by two kinases to uridine triphosphate (UTP) via two sequential reactions with ATP.  First, the diphosphate from UDP is produced, which in turn is phosphorylated to UTP. Both steps are fueled by ATP hydrolysis:
:ATP + UMP → ADP + UDP
:UDP + ATP → UTP + ADP
CTP is subsequently formed by the amination of UTP by the catalytic activity of CTP synthetase. Glutamine is the NH donor and the reaction is fueled by ATP hydrolysis, too:
:UTP + Glutamine + ATP + HO → CTP + ADP + P
Cytidine monophosphate (CMP) is derived from cytidine triphosphate (CTP) with subsequent loss of two phosphates.