Plakoglobin is a member of a protein family with a repeated amino acid motif, the armadillo repeat, and is a cytoplasmic protein found in both adherens junctions and desmosomes. Plakoglobin has been shown to form distinct complexes with cadherins or desmosomal cadherins. Also, plakoglobin has been shown to complex with APC, the tumor suppressor gene product. Recently we isolated a cDNA clone encoding plakoglobin lacking the fourth armdillo repeat of the original 13-repeat protein [Ozawa et al. (1995)J. Biochem. 118, 836–840]. In this study, we established an in vitro assay system to study the molecular interaction of plakoglobin with cadherins, the APC gene product, and α-catenin. Establishment of the system and cloning of an alternate form of plakoglobin cDNA allowed us to examine the biological activity of plakoglobin lacking the fourth armadillo repeat. Experiments with the bacterially expressed 12-repeat plakoglobin revealed that the protein binds to E-cadherin, desmoglein (Dsg2), and APC with lower affinity than the 13-repeat form does. Consistent with the observation that the affinity of α-catenin for these two alternate forms was similar, we found amino acid residues 104 to 145 of plakoglobin, the residues present in both isoforms, are sufficient for its binding to α-catenin.