mtDNA mutations occur in a wide variety of degenerative diseases and cancer. mtDNA seems to be more susceptible to DNA damage and consequently sustains higher rates of mutation than does nuclear DNA (nDNA). Many of the somatic mtDNA mutations in human cancers are located in the displacement loop (D-loop) and in particular in a polycytidine stretch (C-tract) termed D310. The D310 region exhibits polymorphic length variation among individuals and has been described as a "hot spot" for somatic mutations in many cancer types. We used real-time quantitative PCR to analyze mtDNA integrity, damage repair, and induced mutations after exposure of human adult retinal pigment epithelial (ARPE)-19 cells to 4-nitroquinoline 1-oxide, a UV-mimetic and adduct-forming carcinogen, and tert-butyl hydroperoxide, an oxidant. The mtDNA-damage profile depended on the region. Thus, the tRNA coding for glycine (tRNA(G)) was the least affected region, whereas the D-loop, and especially its D310 region, were most sensitive to damage. The time course of repair of mutations of the D-loop and especially the D310 region after exposure to DNA-damaging agents was delayed when compared with other regions and gave rise to common D310 C-tract frame-shift mutations. The induced mutations in the D310 region were predominantly homoplasmic only 7 days after exposure to damage. Our results establish that the D-loop (especially its D310 region) is highly susceptible to mutations because of its vulnerability to DNA damage and inefficient repair mechanisms. Our findings may explain the high frequency of homoplasmic D310 somatic mutations in many tumor types.