Proteolytic degradation of the extracellular matrix bymetastatic tumor cells is initiated by the formation ofinvadopodia, i.e., actin-driven filopodia-like membrane protrusionsendowed with matrix-degradative activity. A signalingcascade involving neural Wiskott-Aldrich syndromeprotein and the Arp2/3actin nucleating complex is involvedin actin assembly at invadopodia. Yet, the mechanism ofinvadopodia formation is poorly understood. Based on theirrole as actin nucleators in cytoskeletal rearrangements,including filopodia formation, we examined the function ofDiaphanous-related formins (DRF) in invadopodia formationand invasion by breast tumor cells. Using small interferingRNA silencing of protein expression in highly invasive MDAMB-231 breast adenocarcinoma cells, we show that threemembers of the DRF family (DRF1–DRF3) are required forinvadopodia formation and two-dimensional matrix proteolysis.We also report that invasion of a three-dimensionalMatrigel matrix involves filopodia-like protrusions enrichedfor invadopodial proteins, including membrane type 1 matrixmetalloproteinase, which depend on DRFs for their formation.These data identify DRFs as critical components of theinvasive apparatus of tumor cells in two-dimensional andthree-dimensional matrices and suggest that different typesof actin nucleators cooperate during the formation of invadopodia.