Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces cell cycle synchronization in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect on the dNTP pools in rho0 cells but not in rho+ cells. In the rho0 cells a 2-3 fold increase in all four deoxynucleotides is observed. Besides from tRNAs and mRNAs the mitochondrial genome is encoding 13 proteins in the electron transport chain, including 2 subunits of the ATP-synthase. As a result of this the rho0 cells have much lower ATP levels than rho+ cells. In order to mimic the ATP situation in rho0 cells the rho+ cells are incubated with the ATP synthase inhibitor, oligomycin. Similar to the rho0 cells the oligomycin incubated rho+ cells display destabilized dNTP pools upon UV irradiation. Our data shows that normal mitochondrial function is prerequisite for retaining stable dNTP pools upon DNA damage. Therefore it is likely that mitochondrial deficiency defects may cause an increase in DNA mutations by disrupting dNTP pool balance.