Human UMP/CMP kinase (cytidylate kinase; EC 2.7.4.14) is responsible for phosphorylation of CMP, UMP, and deoxycytidine monophosphate (dCMP) and also plays an important role in the activation of pyrimidine analogs, some of which are clinically useful anticancer or antiviral drugs. Previous kinetic data using recombinant or highly purified human UMP/CMP kinase showed that dCMP, as well as pyrimidine analog monophosphates, were much poorer substrates than CMP or UMP for this enzyme. This implies that other unidentified mechanisms must be involved to make phosphorylation of dCMP or pyrimidine analog monophosphates inside cells by this enzyme possible. Here, we reevaluated the optimal reaction conditions for human recombinant human UMP/CMP kinase to phosphorylate dCMP and CMP (referred as dCMPK and CMPK activities). We found that ATP and magnesium were important regulators of the kinase activities of this enzyme. Free magnesium enhanced dCMPK activity but inhibited CMPK activity. Free ATP or excess ATP/magnesium, on the other hand, inhibited dCMPK but not CMPK reactions. The differential regulation of dCMPK versus CMPK activities by ATP or magnesium was also seen in other 2'-deoxypyrimidine analog monophosphates (deoxyuridine monophosphate, 5-fluorodeoxyuridine monophosphate, 1-beta-D-arabinofuranosylcytosine monophosphate, and gemcitabine monophosphate) versus their ribose-counterparts (UMP and 5-fluorouridine monophosphate), in a similar manner. The data suggest that the active sites of human UMP/CMP kinase for dCMP and for CMP cannot be identical. Furthermore, enzyme inhibition studies demonstrated that CMP could inhibit dCMP phosphorylation in a noncompetitive manner, with Ki values much higher than its own Km values. We thus propose novel models for the phosphorylation action of human UMP/CMP kinase.