Somatic mutations at a mitochondrial noncoding polycytidine (C)n repeat (polyC) have been associated with tumor progression. We analyzed whether these alterations are due to the inherent mutability of repeated sequences. Insertion and deletion mutations were found in colon (n= 114), stomach (n= 105), endometrium (n= 53), breast (n= 45), lung (n = 35), and prostate (n= 20) tumors. The mutation frequency in colon, gastric, and endometrial tumors was 23, 17, and 11%, respectively, which paralleled the relative extent of microsatellite instability in long mononucleotide repeats observed in tumors with mismatch repair deficiency (colon > stomach > endometrium, relative ratio 10:8:4). Colon tumors with mutations of more than one nucleotide were more advanced in tumor progression. Further, two tumors showing a T > C mutation that restored the homopolymeric repeat, harbored sequential deletion mutations of up to 4 and 6 nucleotides. These results illustrate that the increased mutability of repeated mitochondrial sequences is dependent on the repetitive structure of the DNA molecule and suggest that mutations in the (C)n repeat, whether homoplasmic or not, and by extrapolation, mitochondrial mutations in general, are not the result of selective pressure during tumorigenesis. We also suggest that the (C)n repeat may be used as an universal molecular clock to estimate the relative mitotic history of tumors. © 2006 Wiley-Liss, Inc.