Thymidine kinase 2 (TK2) is one of four deoxynucleoside kinases (dNKs) in humans and plays a crucial role in the initial phosphorylation of pyrimidine nucleosides in the salvage pathway in mitochondria. Nucleoside analogues, like AZT, are substrates of TK2 and induced mitochondrial toxicity in long-term therapy. We found that AZT and FLT inhibited dThd phosphorylation but stimulated dCyd phosphorylation catalyzed by TK2. However, mitochondrial phosphorylation of both dThd and dCyd was inhibited by AZT and FLT. Here a preliminary identification and characterization of mitochondrial factors is reported.