We recently have reported that Wilms' tumor gene WT1 is highly expressed not only in leukemias but also in various types of solid tumors and that WT1 protein is a novel tumor antigen against which cytotoxic T lymphocytes (CTLs) can be elicited by immunization with 9-mer WT1 peptides capable of binding to major histocompatibility complex (MHC) class I molecules. In the present study, plasmid DNA encoding murine full-length WT1 protein was injected intramuscularly into C57BL/6 mice. The mice vaccinated with the WT1 plasmid DNA elicited CTLs against the WT1 protein, and the CTLs specifically killed WT1-expressing tumor cells in a MHC class I-restricted manner. Furthermore, the vaccinated mice rejected the challenges of WT1-expressing tumor cells and survived with no signs of autoimmunity caused by the CTLs. These results demonstrated that vaccination with the WT1 plasmid DNA can elicit CTL responses specific for the WT1 protein, resulting in the acquisition of rejection activity against challenges of WT1-expressing tumor cells. This WT1 DNA vaccination may find clinical application for various types of solid tumors as well as leukemias.