β-Catenin has been identified as an oncogene in colon cancer and melanoma. Phosphorylation of sites in exon 3 of β-catenin leads to degradation of this protein. These sites are primary targets for activating mutations. The frequency with which oncogenic mutations at these sites are found in colorectal cancer is unknown, as is the frequency of their occurrence in other malignancies. We analyzed 92 colorectal cancers (CRCs) and 57 cancer cell lines (representing a diversity of tumor types) to determine the frequency of activating mutations in this gene. Mutations in exon 3 of β-catenin were found in 2 of 92 CRCs and in the colorectal cancer cell line HCT 116. Both tumors with β-catenin mutations exhibited widespread microsatellite instability, which is indicative of a replication error phenotype, a phenotype known to be present in HCT 116. This suggests that mutations in β-catenin are infrequent in CRC and miscellaneous cancer cell lines and may occur in association with a replication error phenotype.