Osteopontin (OPN) is a sialic acid-rich, adhesive, extracellular matrix (ECM) protein with Arg-Gly-Asp cell-binding sequence that interacts with several integrins, including alpha(v)beta(3). Since the ECM is a key regulator of mammary gland morphogenesis, and mammary epithelial cells express OPN at elevated levels, we sought to determine whether this protein plays a role in the postnatal mammary gland development. By generating transgenic mice that express OPN antisense-RNA (AS-OPN mice) in the mammary epithelia we achieved suppression of OPN production in this organ. The pregnant AS-OPN mice displayed a lack of mammary alveolar structures, a drastic reduction in the synthesis of beta-casein, whey acidic milk protein, and lactation deficiency. In agreement with these findings, we uncovered that a mammary cell line, NMuMG, which undergoes both structural and functional differentiation on ECM-coated plates, when transfected with an antisense OPN-cDNA construct, failed to undergo such differentiation. Furthermore, the results of gel-invasion assays demonstrated that these cells manifest elevated matrix metalloproteinase (MMP) activity when OPN expression is significantly reduced. The identity of this proteinase as MMP-2 is confirmed by Western blotting, zymography, and inhibition of its activity by a specific inhibitor, TIMP-2. Taken together, our results demonstrate, for the first time, an essential role of OPN in mammary gland differentiation and that the molecular mechanism(s) of its action, at least in part, involves down-regulation of MMP-2.