Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase type-plasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.