Mitochondrial DNA is more prone to suffering from extensive oxidative damage than nuclear DNA. Base-excision repair which is well established in mitochondria may be involved in the prevention of nucleotides from oxidative damage. It is necessary to consider which severe damage or inadequate repair mainly contributes to the mutations in mtDNA. Human LO₂ liver cells were exposed to 9 mmol/L alloxan for 1 h and then incubated in fresh culture media for 0, 2, 8 and 24 h respectively. The frequencies of oxidative base damage in mtDNA were measured by a quantitative Southern blot coupled with digestion by the enzymes endonuclease Ⅲ and formamidopyrimiding DNA glycosylase. Next, ligation-mediated PCR (LM-PCR) was performed to map damage to specific nucleotides along a ～100 bp fragment including MTTL1 gene. The addition of alloxan to cultured human cells increased the rate of oxidative base damage and ,by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the lesion frequency decreased to levels slightly higher than normal at 8h and returned to normal levels at 24 h. The result of LM-PCR showed 20 hot spots of MTTL1 gene where nucleotides were receiving a majority of damage. The pattern of oxidative damage is like that of point mutation identified in this gene, suggesting the possibility that oxidative damage mainly contributes to the formation of the point mutation in MTTL1 gene.