To investigate differences in gene expression between normal thyroid tissue and papillary thyroid carcinomas, we performed differential display (DD) polymerase chain reaction (PCR) using total RNA from fresh-frozen surgically removed thyroid specimens. Four DD fragments that were overexpressed in tumor tissue were identified as parts of genes from the mitochondrial genome: nicotinamide adenine dinucleotide (NADH) dehydrogenase 5, adenosine triphosphate (ATP) synthase 6, cytochrome b, and cytochrome c oxidase I. The expression profiles of these genes were confirmed by hybridization using a DNA dot-blot array and radioactively labeled complex cDNA probes generated from tumor (30 biopsies) and nontumor (15 biopsies) total RNA. Cytochrome c oxidase III was also found to be overexpressed in papillary carcinomas, while the nuclear-encoded mitochondrial transcription factor A showed similar mRNA expression levels in tumor and nontumor tissue. Electron microscopy showed increased number and size of mitochondria in papillary carcinomas. Immunohistochemistry using a monoclonal antibody recognizing a nuclear-encoded mitochondrial protein showed positivity in all cases of papillary carcinoma (44 samples), while normal thyroid tissue (34 samples) was negative in all cases except 3, in which there was a weak, focal cytoplasmic staining. We conclude that papillary thyroid carcinomas show increased expression of mitochondrial mRNA and proteins, encoded by nuclear as well as mitochondrial genes.