About 15% of sporadic colorectal cancers show microsatellite instability (MSI) due to the inactivation of mismatch repair genes and are termed MSI-H tumors. In these tumors, frameshift mutations in coding repeats have been found within the TGFbeta-RII, BAX, and IGFRII genes that are probably involved in their progression. In the present work, we report frequent mutations in TCF-4, another target gene for instability. TCF-4 codes for a transcription factor that is a crucial member of the adenomatous polyposis coil (APC)/beta-catenin/T-cell factor (TCF) pathway. Fifty percent (4 of 8) of human MSI-H colorectal cell lines and 39% (19 of 49) of MSI-H colorectal primary tumors were found to have a 1-bp deletion in an (A)9 repeat within the coding region of this gene. In contrast, a frameshift mutation was found in only 1 of 56 non-MSI colorectal tumors and in none of 16 non-MSI colorectal cancer cell lines. These results suggest that TCF-4 frameshift mutations are selected for and play a role in colorectal MSI-H tumorigenesis. Depending on different reading frames due to alternatively spliced TCF-4 mRNA, the (A)9 repeat normally codes for several isoforms that could serve as modulators of TCF-4 transcriptional activity. The deletion of one nucleotide in this repeat could change TCF-4 transactivating properties by modifying the respective proportions of the different isoforms.