Factors contributing to the development of thyroid neoplasia remain poorly understood. Recent evidence indicates that overexpression of the inducible cyclooxygenase, COX-2, is important in the pathogenesis of epithelial carcinomas. These studies were undertaken to evaluate whether COX-2 is up-regulated in human thyroid neoplasia. Benign (n = 14), and malignant (n = 14) thyroid nodules were analyzed for expression of COX-2 mRNA by quantitative RT-PCR. Immunoblotting and immunohistochemistry were performed on representative samples. Three human thyroid cancer cell lines were similarly analyzed for COX-2 expression. Levels of COX-2 mRNA were significantly increased in thyroid nodule samples compared with adjacent thyroid tissue in the malignant specimens but not in the benign specimens. Additionally, COX-2 mRNA levels were significantly increased in malignant nodule samples compared with benign nodule samples. COX-2 protein expression was higher in 8 of 10 thyroid nodules compared with the adjacent tissue. Immunohistochemical analysis localized expression of COX-2 to the malignant epithelial cells. Immunofluorescence demonstrated COX-2 protein expression in all three thyroid cell lines. Finally, COX-2 expression could be detected by RT-PCR in fine needle aspiration specimens of thyroid nodules. These data indicate that COX-2 is up-regulated in human thyroid cancer, but not in benign thyroid nodules, and suggest that COX-2 expression may serve as a marker of malignancy in thyroid nodules.