Deoxynucleoside kinases are required for the 5'-phosphorylation of deoxynucleoside analogs used in chemotherapy. Cytoplasmic thymidine kinase (TK1), deoxycytidine kinase (dCK) and mitochondrial thymidine kinase (TK2) were completely purified from human leukemic spleen and their capacities to phosphorylate 43 nucleoside analogs were compared. TK1 showed the most restricted substrate specificity but tolerated 3'-modifications of the sugar ring and some 5-substitutions of the pyrimidine ring. TK2 showed a much broader specificity and phosphorylated pyrimidine bases with bulky 5-substitutions, including cytosine analogs, while sugar analogs with substituents other than OH in the 2' and 3' positions were very poor substrates. dCK showed a very broad specificity phosphorylating several cytosine analogs with 2' and 3' modifications as well as acyclic sugar analogs. Purine deoxyribonucleosides were also efficiently phosphorylated by dCK but in this case sugar modifications led to drastically decreased activity.