Colorectal cancers with microsatellite instability (MSI+ CRCs) caused by dysfunction of DNA mismatch repair have unique clinicopathological characteristics including good prognosis with T-cell infiltration in tumor. To identify tumor antigens that induce immune response against MSI+ CRC, SEREX (serological analysis of recombinant cDNA expression cloning) was applied. By screening a lambda phage cDNA library constructed from three MSI+ CRC cell lines with serum from a patient with MSI+ CRC with abundant T-cell infiltrates in tumor, 64 antigens were isolated. Immunogenicity of each antigen was evaluated by screening sera from patients with various cancers and from healthy individuals, and specific IgG antibodies (Abs) for 49 antigens were detected only in MSI+ CRC patients. A frameshift mutation in the repetitive G sequences (microsatellite) in the coding region of CDX2, one of the identified antigens, was found in the tumor tissue of the patient who had anti-CDX2 serum Ab. The Ab recognized both the COOH-terminal tumor-specific peptides created by the frameshift mutation and the NH(2)-terminal normal peptides of CDX2 when Western blot analysis was performed using various bacterial recombinant CDX2 proteins including the normal and altered peptides, which indicated that immune response could be raised against tumor-specific peptides generated through MSI. The anti-CDX2 Ab was detected only in the patient with the CDX2 frameshift mutation in tumor and disappeared 7 years after the curative resection, suggesting that this immune response may also be useful as a tumor marker. No altered subcellular localization and transcription ability was demonstrated in the mutated CDX2, although decreased expression was suggested in immunohistochemical analysis. Therefore, tumor-specific peptides generated by MSI may be involved in antitumor immune responses and may be useful for the development of diagnostic and therapeutic methods for patients with MSI+ CRC.