The expression levels of the Wilms' tumor gene WT1 were examined in 56 cases of head and neck squamous cell carcinoma (HNSCC) using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). They included 4 cases of floor of mouth, 9 of gingiva, 25 of tongue, 10 of oropharynx, 3 of hypopharynx, and 5 larynx squamous cell carcinoma (SCC). All (100%) of 4 cases of floor of mouth, 5 (56%) of 9 gingiva, 17 (68%) of 25 tongue, 8 (80%) of 10 oropharynx, all (100%) of 3 hypopharynx, and all (100%) of 5 larynx SCC overexpressed the WT1 gene in the range of 3.07 x 10(-4)-8.60 x 10(-1) levels (the WT1 expression level in K562 leukemic cells was defined as 1.0). Thus, 42 (75%) out of 56 cases of HNSCC overexpressed the WT1 gene. The high expression level of the WT1 gene significantly correlated with poor histological tumor differentiation and high tumor stage of HNSCC. Immunohistochemical analysis confirmed the expression of WT1 protein in 6 cases (one floor of mouth, 2 tongue, 2 oropharynx, and one larynx SCC) with overexpression of the WT1 gene. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of 10 exons of the WT1 gene in 5 different HNSCC. These findings suggest an important role of the wild-type WT1 gene in the tumorigenesis of HNSCC.