Mitochondrial DNA (mtDNA) is known for high mutation rates caused by lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of oxygen radicals. We examined the frequency of mutations in the mtDNA D-Loop region in 20 patients with Barrett's carcinoma and associated Barrett's epithelium by automated DNA sequencing. Mutations were detected in eight of 20 (40%) patients in tumor and/or tumor-associated Barrett's epithelium. In six of eight positive cases, the mutations were detected only in the tumor, one of eight showed mutations in tumor and Barrett's epithelium, and one of eight only in Barrett's epithelium. The degree of dysplasia in Barrett's epithelium was classified low-grade in one and high grade in the two specimens. There was no association of mtDNA D-Loop mutations with histopathological stage of disease or tumor grading. We present the first study of frequent occurrence of mutations in the mtDNA D-Loop regions in adenocarcinomas in Barrett's esophagus. Furthermore, this study supports the hypothesis that oxidative damage might be a mechanism for the induction of adenocarcinoma in Barrett's esophagus. Since mutations were identified in tumor-associated dysplastic Barrett's epithelium, they also might become a marker for the malignant potential of Barrett's epithelium.