Mutation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates the majority of colorectal (CR) cancers. One consequence of this inactivation is constitutive activation of beta-catenin/Tcf-mediated transcription. To further explore the role of the APC/beta-catenin/Tcf pathway in CR tumorigenesis, we searched for mutations in genes implicated in this pathway in CR tumors lacking APC mutations. No mutations of the gamma-catenin (CTNNG1), GSK-3alpha (GSK3A), or GSK-3beta (GSK3B) genes were detected. In contrast, mutations in the NH2-terminal regulatory domain of beta-catenin (CTNNB1) were found in 13 of 27 (48%) CR tumors lacking APC mutations. Mutations in the beta-catenin regulatory domain and APC were observed to be mutually exclusive, consistent with their equivalent effects on beta-catenin stability and Tcf transactivation. In addition, we found that CTNNB1 mutations can occur in the early, adenomatous stage of CR neoplasia, as has been observed previously with APC mutations. These results suggest that CTNNB1 mutations can uniquely substitute for APC mutations in CR tumors and that beta-catenin signaling plays a critical role in CR tumorigenesis.