The response of endothelial cells to angiogenic stimuli has been shown to be influenced by the extracellular microenvironment. We tested whether thrombospondin, an extracellular matrix protein, modulated the spontaneous formation of cords by endothelial cells in vitro. Despite continued proliferation, a decrease in secreted thrombospondin was detected in cord-containing, as compared with subconfluent, cultures of both aortic and microvascular endothelial cells. Consistent with this trend, mRNA levels of thrombospondin decreased by factors of 16 in aortic and 60 in microvascular cultures that contained endothelial cords. Since thrombospondin was immunolocalized to fibrillar arrays that appeared to be associated with endothelial cords, we added anti-thrombospondin IgG to cord-forming cultures to limit the availability of the protein during this process. In the presence of anti-thrombospondin antibodies, there was a 33-50% increase in cord formation. These results suggest that thrombospondin is an inhibitor of angiogenesis in vitro and are consistent with its proposed roles as a destabilizer of endothelial cell focal contacts and as an inhibitor of endothelial cell proliferation.