Vascular basement membrane is an important structural component of blood vessels and has been shown to interact with and modulate vascular endothelial behavior during angiogenesis. During the inductive phase of tumor angiogenesis, this membrane undergoes many degradative and structural changes and reorganizes to a native state around newly formed capillaries in the resolution phase. Such matrix changes are potentially associated with molecular modifications that include expression of matrix gene products coupled with conformational changes,which expose cryptic protein modules for interaction with the vascular endothelium. We speculate that these interactions provide important endogenous angiogenic and anti-angiogenic cues. In this report, we identify an important anti-angiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of theα ₁ chain of type IV collagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and 293 embryonic kidney cells. We demonstrate that arresten functions as an anti-angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation,and Matrigel neovascularization. Arresten inhibits the growth of two human xenograft tumors in nude mice and the development of tumor metastases. Additionally, we show that the anti-angiogenic activity of arresten is potentially mediated via mechanisms involving cell surface proteoglycans and the α₁β₁ integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.