To define the unique contributions of the α subunit cytoplasmic tails of the α1β1 and α2β1 integrin to epithelial differentiation and branching morphogenesis, a variant NMuMG cell line lacking α1β1 and α2β1 integrin expression was stably transfected with the full-length α2 integrin subunit cDNA (X2C2), chimeric cDNA consisting of the extracellular and transmembrane domains of the α2 subunit and the cytoplasmic domain of the α1 subunit (X2C1), or α2 cDNA truncated after the GFFKR sequence (X2C0). The X2C2 and X2C1 transfectants effectively adhered, spread, and formed focal adhesion complexes on type I collagen matrices. The X2C0 transfectants were less adherent to low concentrations of type I collagen, spread less well, and formed poorly defined focal adhesion complexes in comparison to the X2C2 and X2C1 transfectants. The X2C2 and X2C1 transfectants but not the X2C0 transfectants proliferated on collagen substrates. Only the X2C2 transfectants developed elongate branches and tubules in three-dimensional collagen gels and migrated on type I collagen. These findings suggest a unique role for the α2 integrin cytoplasmic domain in postligand binding events and cooperative interactions with growth factors that mediate epithelial differentiation and branching morphogenesis. Either intact α1 or α2 integrin subunit cytoplasmic domain can promote cell cycle progression.