It has been established that the invasive behavior of cancer cells can be regulated by alterations in their extracellular environment. We investigated whether extracellular matrix isolated from nulliparous and postlactating (involuting) rat mammary glands differentially modulated the metastatic behavior of human breast cancer cells. Using modified Boyden chamber and three-dimensional culture assays, nulliparous mammary matrix was found to suppress motility and invasion in highly metastatic MDA-MB-435 cells, whereas involution mammary matrix supported motility and invasion in highly metastatic MDA-MB-435 cells,but not in cells with low metastatic potential. Biochemical characterization of the matrices revealed intact fibronectin (FN) and low matrix metalloproteinase activity in nulliparous mammary matrix and fragmented FN and high matrix metalloproteinase activity in the matrix isolated from involuting glands. Purified intact FN was found to inhibit cell invasiveness, whereas FN fragments enhanced cell invasiveness in a matrix metalloproteinase-dependent manner. These data suggest that physiological changes that occur in the mammary extracellular matrix as a result of reproductive status alter the in vitro parameters of metastatic potential.