Although integrins are known to mediate adhesive binding of cells to the extracellular matrix, their role in mediating cellular growth, morphology, and differentiation is less clear. To determine more directly the role of the α₂β₁ integrin, a collagen and laminin receptor, in mediating the collagen-dependent differentiation of mammary cells, we reduced expression of the integrin by the well differentiated human breast carcinoma cell line, T47D, by stably expressing α₂ integrin antisense mRNA. Flow cytometry demonstrated that the antisense-expressing clones had levels of α₂β₁ integrin on their surfaces that were decreased by 30-70%. Adhesion of antisense-expressing clones to both collagens I and IV was decreased relative to controls in a manner that correlated with the level of cell surface α₂β₁ integrin expression. Adhesion to fibronectin and laminin were not affected. Motility across collagen-coated filters in haptotaxis assays was increased for only those clones that exhibited intermediate levels of adhesion to collagen, suggesting that an intermediate density of cell-surface α₂β₁ integrin optimally supports cell motility. When cultured in three-dimensional collagen gels, T47D cells organized in a manner suggestive of a glandular epithelium. In contrast, antisense-expressing clones with decreased α₂β₁ integrin were not able to organize in three-dimensional collagen gels. The growth rate of T47D cells was reduced when the cells were cultured in three-dimensional collagen gels. Unlike adhesion, motility, and morphogenesis, growth rates were unaffected by reduction of α₂β₁ integrin expression. Our results suggest that adhesive interactions mediated by a critical level of surface α₂β₁ integrin expression are key determinants of the collagen-dependent morphogenetic capacity of mammary epithelial cells.