Studies of Wnt activation in gastric cancer have yielded conflicting results. The goals of this study were to determine the frequency of Wnt pathway activation and beta-catenin mutation in these tumors. Three hundred eleven gastric cancers were examined for beta-catenin expression by immunostaining and dissected using laser capture microscopy to obtain DNA from those tumors with nuclear beta-catenin. Exon 3 of beta-catenin was amplified using PCR and sequenced. Ninety gastric cancers (29%) displayed nuclear beta-catenin. DNAs from 73 tumors were amplified and sequenced; 19 (26%) contained mutations in exon 3 of beta-catenin, whereas no mutations were detected in 19 tumors negative for beta-catenin nuclear staining (P < 0.05). Most mutations were adjacent to or abolished known regulatory phosphorylation sites. Mutations in exon 3 of beta-catenin are common in gastric cancer that display nuclear beta-catenin. These results suggest that Wnt pathway activation contributes to carcinogenesis in a subset of gastric adenocarcinomas.