β-catenin is a multifunctional protein found in three cell compartments: the plasma membrane, the cytoplasm and the nucleus. The cell has developed elaborate ways of regulating the level and localization of β-catenin to assure its specific function in each compartment. One aspect of this regulation is inherent in the structural organization of β-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, we found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with β-catenin; the association of β-catenin with LEF-1 was competed out by the E-cadherin cytoplasmic domain. Similarly, LEF-1 and adenomatous polyposis coli (APC) formed separate, mutually exclusive complexes with β-catenin. In Wnt-1-transfected C57MG cells, free β-catenin accumulated and was able to associate with LEF-1. The absence of E-cadherin in E-cadherin−/− embryonic stem (ES) cells also led to an accumulation of free β-catenin and its association with LEF-1, thereby mimicking Wnt signaling. β-catenin/LEF-1-mediated transactivation in these cells was antagonized by transient expression of wild-type E-cadherin, but not of E-cadherin lacking the β-catenin binding site. The potent ability of E-cadherin to recruit β-catenin to the cell membrane and prevent its nuclear localization and transactivation was also demonstrated using SW480 colon carcinoma cells.