Activation of the macrophage colony-stimulating factor receptor (CSF-1R) by its cognate ligand CSF-1 dramatically increases the tumorigenicity and invasive potential of both normal and neoplastic mammary epithelial cells. Recent studies have suggested that the Ets2 transcription factor plays a central role in mediating CSF-1R-induced mitogenesis in fibroblasts. To determine whether the Ets2 transcription factor can also mediate CSF-1- and CSF-1R-stimulated signaling pathways in mammary epithelial cells, we expressed a dominant negative mutant, Ets2, in the CSF-1R- and Ets2-positive BT20 breast carcinoma cell line and examined its effects on CSF-1-induced cellular invasion and on colony formation in soft agar. Our data show that stable expression of the mutant Ets2 in BT20 cells completely inhibits the formation of soft agar colonies and abolishes the CSF-1-stimulated invasion of these cells through a barrier of reconstituted basement membrane (Matrigel). We have also demonstrated that the expression of this Ets2 mutant is capable of interrupting the CSF-1R-regulated intracellular signaling pathways by inhibiting CSF-1-induced c-myc, c-fos, and c-jun expression in BT20 cells. Our results are the first demonstration of an important role for the Ets2 transcription factor in the regulation of the anchorage-independent growth and cellular invasiveness of neoplastic mammary epithelial cells.