We identified that whirlin is localized to chromosome 9q32-33, and is up-regulated in colorectal cancer tissues by using oligonucleotide array techniques and the Sosui system (http://www.tuat.ac.jp/~mitaku/sosui/). The deduced 920-amino acid protein encoded by the whirlin gene contains three PDZ domains and a proline-rich region that separates PDZ2 from PDZ3, which is located at the C terminus. As previously reported, human whirlin gene is alternatively spliced to form a long and a short transcript in situ hybridization. The sequence of the encoded protein shows that the short C-terminal isoform contains one PDZ domain and the proline-rich domain (whirlin isoform 2), whereas the long isoform is composed of all three PDZ domains and the proline-rich domain (whirlin isoform 1). The gene expression of whirlin was found to be up-regulated in colorectal cancer tissues compared with matched normal colon tissues by semi-quantitative RT-PCR (P<0.05). Western blotting detected whirlin protein with a molecular mass of 49.3 kDa in colorectal cancer samples, suggesting that the whirlin protein overexpressed in colorectal cancer samples is the short C-terminal isoform 2. Its expression was recognized in colorectal cancer cell lines and was increased in accordance with tumor progression in colorectal cancer patients. Immunohistochemistry showed high levels of staining for whirlin isoform 2 only in the mucosal glands in colon cancers, but this protein was barely detected in normal colonic glands. Immunoelectron microscopic findings showed that whirlin isoform 2 is localized on plasma membranes and endoplasmic reticulum membranes, but not in the nuclei. Tissue microarrays showed that whirlin isoform 2 is abundantly expressed in colon cancers with lymph node metastasis compared with those without lymph node metastasis, and overexpression of this protein was associated with tumor progression. In conclusion, we demonstrated that whirlin isoform 2 is highly expressed in colon cancer tissues and that it is related to tumor progression.