Ca²⁺-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include α-, β-, and γ-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either β- or γ-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and β-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of γ-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and β-catenin co-immunoprecipitate with E-cadherin. Laserscanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with γ-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for of the γ-catenin signal. Two-dimensional gel electrophoresis resolved the β-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI ∼ 6.1). The other protein comigrated with γ-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of ∼4.25; we refer to this protein as γ-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains α-, β-, and γ-catenin. Although plakoglobin shares sequence similarity with β-catenin, and comigrates with γ-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The asociation of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.