The mammalian deoxyribonucleoside kinases are deoxycytidine kinase, thymidine kinase 1 and 2 and deoxyguanosine kinase. These enzymes phosphorylate deoxyribonucleosides and thereby provide an alternative to de novo synthesis of DNA precursors. Their activities are essential for the activation of several chemotherapeutically important nucleoside analogues. In recent years, these enzymes have been thoroughly characterised with regard to structure, substrate specificity and patterns of expression. In this review, these results are reviewed and furthermore, the physiologic metabolic role of the anabolic enzymes is discussed in relation to catabolic pathways. The significance of this information for the development of therapeutic protocols and choice of animal model systems is discussed. Finally, alternative pathways for nucleoside analogue phosphorylation are surveyed, such as the phosphotransfer capacity of 5′-nucleotidase.