Multiple somatic mitochondrial DNA mutations are frequently reported in human tumors, but the process leading to homoplasmic transformation and accumulation of multiple mutations in the same tumor cell lineage remains a mystery. We address possible mechanisms responsible for the generation of multiple mitochondrial (mt)DNA mutations observed in a high frequency of prostate tumors using sensitive mutant-specific PCR coupled with laser capture microdissection. Analysis of prostate tumors with multiple mtDNA mutations in the control region indicates that the mutations are locally confined, that the multiple mutations exist on the same molecules and that more than one mtDNA mutant species co-exists in the same neoplastic lesion. These results suggest an unusually rapid process in mtDNA mutagenesis during tumor progression. On the basis of prostate tumor cell kinetics, we propose a unique process of mitochondrial hyper-mutagenesis, probably mediated by cellular oxidative stress, to account for a burst of multiple mtDNA mutations in human prostate tumors.