The effects of lactogenic hormones on urokinase plasminogen activator (u-PA) produced by bovine mammary epithelial cells (MAC-T) were examined. High levels of u-PA activity were detected in growth arrested cells cultured on plastic. This suggests that high levels of PA activity alone are not sufficient to induce proliferation of bovine mammary epithelial cells. Cells were cultured on various extracellular matrices: plastic, fibronectin, collagen, matrigel, and laminin. Basal levels of u-PA activity in media from MAC-T cells cultured on matrigel were 1.6-, 2-, 2- and 3.5-fold higher than that of cells cultured on plastic, fibronectin, collagen, and laminin, respectively. Insulin increased (P<0.01) mammary epithelial u-PA activity on a per cell basis, an effect observed irrespectively of the type of extracellular matrix onto which cells were cultured. Not unexpectedly, insulin-like growth factor (IGF)-I, Des(1-3) IGF-I and IGF-II increased mammary epithelial u-PA activity on a per cell basis and u-PA mRNA levels, thus, mimicking the effect of insulin. Dexamethasone suppressed (P<0.01) u-PA activity but was unable to suppress the insulin-induced increase in u-PA activity of cells cultured on various extracellular matrices. These data indicate that u-PA activity is modulated by both lactogenic hormones and the type of extracellular matrix.