Mitochondrial deoxyguanosine kinase (dGK) (EC 2.7.1.113) was purified to apparent homogeneity from bovine brain. The molecular mass of the native protein was 56 kDa, as judged by gel filtration, and one single band of 28 kDa was seen in sodium dodecyl sulfate-gel electrophoresis. 2'-Deoxyguanosine (dGuo) (Km, 7.6 microM), 2'-deoxyinosine, and 2'-deoxyadenosine (Km, 60 microM) were substrates for the enzyme as well as several dGuo analogs containing a lipophilic substituent at C-2'. Carbocyclic dGuo, 9-beta-D-arabinofuranosylguanine, 9-beta-D-arabinofuranosylhypoxanthine, and 9-beta-D-arabinofuranosyladenine were substrates for the enzyme, whereas no 3'-modified dGuo analogs were effective. Interestingly, 2-chloro-2'-deoxyadenosine (CdA) was found to be an efficient substrate for dGK (Km, 85 microM). Subcellular fractionation of human CEM lymphoblasts showed that extracts of mitochondria contain significant CdA phosphorylating activity (71.5 pmol/mg/min) that is not inhibited by excess of 2'-deoxycytidine (dCyd). This contrasts with the CdA phosphorylating activity found in cytosolic extracts, which is carried out by dCyd kinase and strongly inhibited by excess of dCyd. The efficient CdA phosphorylation by mitochondrial dGK is a novel finding that may have far reaching implications for the clinical use of this potent cytostatic drug.