Background:The ability of TMP to serve as a precursor of mitochondrial synthesis of TTP is not well understood. Results: TMP cannot be converted to TTP except by breakdown to thymidine in isolated mitochondria. Conclusion: Thymidine is the sole source for TTP synthesis in the mitochondrial matrix. Significance: Thymidine salvage in mitochondria is crucial to understand mitochondrial DNA depletion diseases caused by mitochondrial thymidine kinase (TK2) deficiency.