In the endometrium, angiogenesis is a physiological process, whereas in most adult tissues neovascularization is initiated only during tissue repair or pathological conditions. Pericellular proteolysis plays an important role in angiogenesis being required for endothelial cell migration, invasion, and tube formation. We studied the expression of proteases by human endometrial microvascular endothelial cells (hEMVECs) and their involvement in the formation of capillary tubes and compared these requirements with those of foreskin MVECs (hFMVECs). Inhibition of urokinase and matrix metalloproteinase (MMP) both reduced tube formation in a fibrin or fibrin/collagen matrix. hEMVECs expressed various MMP mRNAs and proteins; in particular MMP-1, MMP-2, and membrane-type (MT)1-, MT3-, and MT4-MMPs. MT3- and MT4-MMP mRNA expressions were significantly higher in hEMVECs than in hFMVECs. Other MT-MMP mRNAs and MMP-9 were hardly detectable. Immunohistochemistry confirmed the presence of MT3-MMP in endothelial cells of endometrial tissue. Overexpression of tissue inhibitor of MMP (TIMP)-1 or TIMP-3 by adenoviral transduction of hEMVECs reduced tube formation to the same extent, whereas only TIMP-3 was able to inhibit tube formation by hFMVECs. Tube formation by hEMVECs was partly inhibited by the presence of anti-MT3-MMP IgG. Thus, in contrast to tube formation by hFMVECs, which largely depends on MT1-MMP, capillary-like tube formation by hEMVECs is, at least in part, regulated by MT3-MMP. Chemicals / CAS: collagen, 9007-34-5; fibrin, 9001-31-4; gelatinase A, 146480-35-5; immunoglobulin G, 97794-27-9; interstitial collagenase, 9001-12-1; tissue inhibitor of metalloproteinase 1, 140208-24-8; tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; urokinase, 139639-24-0; Collagen Type I; Matrix Metalloproteinase 16, EC 3.4.24.-; Matrix Metalloproteinases, Membrane-Associated, EC 3.4.24.-; Metalloendopeptidases, EC 3.4.24.-; MMP16 protein, human; Plasmin, EC 3.4.21.7; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-3; Urinary Plasminogen Activator, EC 3.4.21.73