Mitochondrial thymidine kinase (TK2) phosphorylates pyrimidine nucleosides to monophosphates and is expressed constitutively through the cell cycle in all cells. Because of the overlap of its substrate specificity with that of the cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), it has been difficult to determine the role of TK2 in activating nucleosides used in chemotherapy. In this report, we described the construction of a recombinant Escherichia coli strain which could be used to test if TK2 activity is limiting for the toxicity of nucleosides. Enzymes of bacterial origin which are involved in thymidine and deoxyuridine anabolism and catabolism were eliminated, and the cDNA for human TK2 was introduced. In the crude extract of the engineered E. coli, the level of thymidine kinase was, after induction of TK2 expression, several hundred fold higher than in the control strain. Several pharmacologically interesting nucleoside analogues, including 3'-azidothymidine, 2',3'-didehydro-2',3'-dideoxythymidine, and 2', 3'-dideoxy-beta-L-3'-thiacytidine, were tested for their effects on the growth of this recombinant strain. For a comparison, the phosphorylation of these compounds was determined with purified recombinant TK1, TK2, and dCK. A correlation was observed between the phosphorylation of several of these compounds by TK2 and their effects on bacterial growth. These results demonstrate that activation of growth-inhibiting pyrimidine nucleosides can be catalyzed by TK2, and together with recombinant E. coli strains expressing other cellular nucleoside kinases, this whole-cell bacterial system may serve as a tool to predict the efficacy and side effects of chemotherapeutic nucleosides.