beta-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although beta-catenin has been shown to participate in many protein-protein interactions, it is not clear which combinations of beta-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular beta-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of beta-catenin, alpha-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell-cell adhesion. APC is also essential for N-terminal phosphorylation of beta-catenin within this complex. Each component of beta-catenin/APC/alpha-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential beta-catenin-interacting proteins, and define when and where a specific complex forms.