Oxidative stress is considered to be one of the most important phenomena involved in the process of aging and age-related diseases. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) has been frequently used as a marker for oxidative stress. However, the origin of extracellular 8-oxo-dG is not well understood. The aim of this work was to investigate the nucleotide pool and the role of the human mutT homologue protein (hMTH1) in the appearance of extracellular 8-oxo-dG in a cellular model system. For this purpose we used primary human fibroblast cells, which were transfected by siRNAs homologous to hMTH1. Extracellular 8-oxo-dG in cell culture media after exposure of the cells to ionizing radiation was measured as enzyme-linked immunosorbent assay reactivity. Our results demonstrate the profound effect of both hMTH1 expression and nucleotide pool size on the cellular excretion of 8-oxo-dG, suggesting that the nucleotide pool is a significant target for the formation of extracellular 8-oxo-dG.