To begin to determine the role of the alpha 2 beta 1 integrin and its ligands, collagen I, collagen IV, and laminin, in mammary epithelial differentiation in vivo, we determined the expression of these molecules by in situ hybridization and immunofluorescence in the developing mouse mammary gland. Expression of collagen I, collagen IV, and laminin mRNAs in the mammary gland during puberty corresponded to the period of greatest growth of the gland, 4-7 weeks postnatally. Collagen I expression preceded collagen IV expression, both of which preceded laminin expression, suggesting an important temporal sequence of extracellular matrix (ECM) production. When growth of the epithelium ceased in the adult virgin gland, expression of all three mRNAs became undetectable. Following the onset of pregnancy these molecules were re-expressed with the same chronology observed during puberty. Collagen I, collagen IV, and laminin were expressed by stromal cells immediately surrounding the developing ductal epithelium. Surprisingly, we found no expression of ECM components in the epithelial cells, suggesting the mammary epithelium does not synthesize its own basement membrane. The distribution of collagen I was consistent with a role in duct formation, since collagen I was strikingly abundant around larger mammary ducts, but was sparse around growing endbuds or alveoli. Conversely, there was abundant laminin near growing endbuds and around alveoli, and less around large ducts, suggesting its role is different than collagen I. The alpha 2 beta 1 integrin was present on the basal, lateral, and apical surfaces of the mammary epithelium throughout postnatal development and pregnancy. The alpha 2 beta 1 integrin expression was strongest at midpregnancy, suggesting a role for alpha 2 beta 1 integrin in the alveolar formation that occurs at this time. The alpha 2 beta 1 integrin expression decreased dramatically in the lactating gland. Our results suggest that alpha 2 beta 1 integrin interactions with its temporally and spatially regulated ligands, collagen I, collagen IV, and laminin, could play an important role in mammary morphogenesis in vivo.