A cDNA encoding mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) was cloned from a mouse T-cell lambda ZAP cDNA library. An insert of 2.8 kilobases (kb) contained the entire coding sequence of 780 base pairs. The protein coding sequence was 88% homologous at the nucleotide level with human dCK cDNA (Chottiner, E. G., Shewach, D. S., Datta, N. S., Ashcraft, E., Gribbin, D., Ginsburg, D., Fox, I. H., and Mitchell, B. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1531-1535). At the amino acid level the homology was greater with only 16 of the 260 amino acids being different. Northern blot analyses revealed a size of 3.4 kb for mouse dCK mRNA as compared with 2.8 kb for human dCK. Part of the 3'-untranslated region was conserved between human and mouse dCK cDNA in contrast to the remainder of the 3'-sequence which was unrelated and about 500 nucleotides longer in mouse dCK cDNA. Mouse dCK cDNA showed cross-hybridization with several bands in EcoRI-digested genomic DNA from seven different mammalian species and chicken but not with yeast DNA. Both mouse and human dCK were cloned into the T5 promotor pQE30 vector system, expressed in Escherichia coli and purified to homogeneity. The kinetic constants for dCyd phosphorylation were similar for the human and mouse enzymes and also similar to what previously has been observed for dCK purified from human tissues. Mouse dCK was less efficient with regard to dAdo, dGuo, and ddCyd phosphorylation as compared with human dCK when using ATP as phosphate donor in a phosphoryl transfer assay.