A facile and fast approach for the purification of deoxycytidine kinase (dCK) from calf thymus was developed using a fast performance liquid chromatography system. A 73-fold enrichment of the enzyme was noted compared to unfractionated dCK. Characterization studies demonstrated that dCK had a molecular mass of 31 kDa using SDS-PAGE, an optimum pH of 7.0 and the enzyme maintained stability between 30 and 40°C. The rapid preparation of dCK demonstrated here will be valuable in the synthesis of nucleotide analogs.