Three key enzymes in the anabolic phosphorylation of deoxyribonucleosides and deoxyribonucleoside analogs were purified i.e. cytoplasmic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and cytoplasmic deoxycytidine kinase (dCK) from human, mouse and monkey liver and spleen. Their subunit structure and substrate specificities were compared. Extensive purification of TK1 and dCK from mouse spleen and TK2 from mouse and monkey livers revealed major polypeptide bands of 25, 30 and 28 kD, respectively, on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which are very similar to the subunit molecular weights of the corresponding human enzymes. Affinity purified polyclonal antibodies against human dCK also cross-reacted with 30 kD bands in extracts from both mouse and monkey spleen. Thus, the molecular weights of the subunits of these three enzymes appeared to be very similar in all three species. TK1 and TK2 from these different sources appeared to have similar substrate specificities against several deoxyribonucleoside analogs. However, mouse dCK differed significantly from monkey and human dCK in its capacity to phosphorylate dAdo and 2',3'-dideoxycytidine (ddCyd) with a Vmax approximately 10-fold lower than that of the two latter enzymes. The Km and Vmax values for dCyd and arabinocytosine appeared to be very similar with the enzymes from all three species. The fact that mouse dCK shows low activity with dAdo and ddCyd explains differences reported previously in the metabolism of dAdo and ddCyd in mouse compared to that in human lymphocytes. These results argue against the use of mice as model systems for human deoxynucleoside metabolism.