The expression levels of the Wilms' tumor gene WT1 were examined in 34 primary thyroid cancers (24 papillary, 5 follicular, 1 anaplastic, and 4 medullary carcinomas), 17 thyroid follicular adenomas, and 6 normal-appearing thyroid tissues using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In 33 of 34 thyroid cancers, the WT1 mRNA was expressed at levels ranging from 5.0 x 10 (-5) to 8.3 x 10 (-2) levels (WT1 expression level in K562 leukemic cells was defined as 1.0). The WT1 mRNA expression levels were significantly higher than those in either thyroid follicular adenomas (P < 0.001) or normal-appearing thyroid tissues (P < 0.01). Immunohistochemical analysis confirmed the expression of WT1 protein in 20 of 21 thyroid cancers with WT1 mRNA expression. WT1 protein was also detected in 6 of 7 follicular adenomas with WT1 mRNA expression. However, the intensity of staining of WT1 protein in adenoma cells was weaker than that in cancer cells and its expression was restricted to approximately 30-80% of adenoma cells in the tumors examined. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of the 10 exons of the WT1 gene in all of the 9 different thyroid cancers. These findings indicate an important role of the wild-type WT1 gene in the tumorigenesis of primary thyroid cancer.