Mammalian mitochondrial deoxyguanosine kinase (dGK) is responsible for phosphorylation of purine deoxyribonucleosides in the mitochondrial matrix. Using a RT-PCR-generated probe, based on amino acid sequence information from proteolytic fragments of purified bovine dGK, we have cloned a cDNA from a human brain cDNA library that encodes a 30 kDa protein. The deduced amino acid sequence of this protein included the sequence of all six peptides isolated and sequenced from purified dGK. Expression and purification of recombinant protein from induced Escherichia coli extracts revealed that it catalyses efficient phosphorylation of dGuo, arabinosyl guanine, dAdo, 2-chloro-2'-deoxyadenosine and dIno similar to purified dGK. Northern blot analysis demonstrated one dominant positive mRNA of 1.35 kb and it was found in several tissues at similar levels. The coding sequence of dGK showed 46% identity to the coding sequence of cytosolic deoxycytidine kinase, and conserved sequence motifs among the known deoxynucleoside kinase were identified.