['To determine the timing of mitochondrial mutations in the progression of head and neck squamous cell carcinoma.', 'Twenty-three mitochondrial mutations were identified in 12 tumors using a high-throughput mitochondrial sequencing array. Areas of adjacent dysplastic and normal epithelium adjacent to tumors were sequenced using conventional methods for the presence of mutations that occurred in the corresponding tumor.', 'Two of 23 (8.7%) tumor mitochondrial mutations (2 of 12 tumors) were present in both the areas of adjacent dysplasia and normal epithelium. Five of 23 (21.7%) tumor mitochondrial mutations (4 of 12 tumors) were present in areas of adjacent dysplasia. Eleven of 12 tumors contained nonsynonymous mutations that resulted in protein coding alterations. A significant difference (P < 0.01, chi(2)) was found in the incidence of mitochondrial mutation that occurred after development of cancer compared with adjacent areas dysplasia and normal epithelium.'] Are mitochondrial mutations a late event in the progression of head and neck squamous cell cancer?