Deoxycytidine kinase is a key anabolic enzyme for the activation of ara-C and other antitumor drugs, as well as normal purine and pyrimidine deoxynucleotides. Previously, two forms of the kinase have been identified; deoxycytidine kinase I (70 kDa) and deoxycytidine kinase II (70 kDa). Deoxycytidine kinase I utilized dCyd and ara-C as substrates, while deoxycytidine kinase II used dCyd and dThd as substrates. Deoxycytidine kinase kinase II had very low activity on ara-C as a substrate. We report a procedure for the purification of a novel deoxycytidine kinase (52 kDa) from isolated human peripheral blood leukemia cell mitochondria. This enzyme has activity similar to deoxycytidine kinase II. The enzyme was extracted from the mitochondria with digitonin (1 mg/8 mg protein) and 0.3 M NaCl, and the extract was purified by DEAE-cellulose chromatography and thymidine-Sepharose affinity chromatography. This procedure produced a near homogeneous enzyme preparation with a yield of 70%. The mitochondrial deoxycytidine kinase was localized to the outer mitochondrial membrane. The enzyme phosphorylated dCyd (Km = 17 microM), however, ara-C was not a good substrate for the mitochondrial deoxycytidine kinase. ATP was the best phosphate donor, whereas dCTP and dTTP were potent inhibitors of mitochondrial deoxycytidine kinase. In contrast, phosphorylation of ara-C by deoxycytidine kinase I utilized GTP, dGTP, or ATP as a phosphate donor.