alpha-Catenin is an intracellular protein that associates with the carboxy-terminal region of cadherin, a cell adhesion molecule, via beta-catenin or gamma-catenin (plakoglobin). Linkage of cadherin to the cytoskeleton by catenins is required for full cadherin activity. Following transfection of an alpha-catenin-deficient colon carcinoma cell line with a series of alpha-catenin constructs, we discovered that the restoration of alpha-catenin expression results in reduced proliferation in three-dimensional multicellular spheroids, but not in two-dimensional monolayer cultures. The cellular function of alpha-catenin has not been compared between cells in three- and two-dimensional culture; this is the first evidence that growth regulation in three-dimensional cultures requires signaling mediated by alpha-catenin. Two classes of constructs, containing deletions in either the central segment or the COOH terminus of the molecule, both induced morphological changes, including cell compaction, and suppressed cell growth in three-dimensional cultures. In alpha-catenin-expressing cells, inhibition of cadherin cell adhesion by treatment with anti-E-cadherin antibodies resulted in a similar phenotype as that observed following the loss of alpha-catenin. Therefore, both the homophilic interaction of the cadherin extracellular domain and the linkage of the cadherin cytoplasmic domain to the actin cytoskeleton by alpha-catenin are necessary for growth control in three-dimensional culture.