In vitro studies have shown that Drosophila melanogaster has a highly efficient single deoxyribonucleoside kinase (dNK) multisubstrate enzyme. dNK is related to the mammalian Thymidine Kinase 2 (TK2) group involved in the nucleotide synthesis salvage pathway. To study the dNK function in vivo, we constructed transgenic Drosophila strains and impaired the nucleotide de novo synthesis pathway, using antifolates such as aminopterin. Our results show that dNK overexpression rescues both cell death and cell cycle arrest triggered by this anti-cancer drug, and confers global resistance on the fly. Moreover, we show that fly viability and growth depend on the exquisite ratio between dNK expression and its substrate thymidine (dT) in the medium, and that increased dT concentrations trigger apoptosis and a decrease in body mass when dNK is mis-expressed. Finally, dNK expression, unlike that of TK2, is cell cycle dependent and under the control of CyclinE and the dE2F1 transcription factor involved in the G1/S transition. dNK is therefore functionally more closely related to mammalian TK1 than to TK2. This strongly suggests that dNK plays a role in cell proliferation in physiological conditions.