Although the WT1 gene was originally isolated as a tumor suppressor gene from Wilms' tumor, oncogenic roles for WT1 have been reported in several tumors. Here, we present new findings of high levels of WT1 expression associated with the suppression of lymph node metastasis in patients with human lung squamous cell carcinoma (SCC). We investigated the effect of down-regulated WT1 gene expression on the invasive phenotype of the SCC cell line RERF-LC-AI. Invasive ability was enhanced in WT1-specific siRNA-transfected cells, and a WT1 target gene p21(Waf1/Cip1) was isolated by comprehensive gene expression analysis. As several isoforms are produced from the WT1 gene, we isolated eight major WT1 isoforms from a cDNA library and cloned each variant into an expression vector. Luciferase reporter assays revealed that p21(Waf1/Cip1) expression was enhanced only by the WT1 cDNA variants that included a three-amino acid deletion (-KTS). Our results suggested that the -KTS-containing variants of WT1 are directly involved in the regulation of p21(Waf1/Cip1) expression and the subsequent suppression of lymph node metastasis in human lung squamous cell carcinoma.