Thrombospondin 1 (TSP1) inhibits angiogenesis and modulates endothelial cell adhesion, motility, and growth. The antiproliferative activity of TSP1 is mimicked by synthetic peptides derived from the type I repeats of TSP1 that antagonize fibroblast growth factor 2 and activate latent transforming growth factor beta. These TSP1 analogues induced programmed cell death in bovine aortic endothelial cells based on morphological changes, assessment of DNA fragmentation, and internucleosomal DNA cleavage. Intact TSP1 also induced DNA fragmentation. The endothelial cell response was specific because no DNA fragmentation was induced in MDA-MB-435S breast carcinoma cells, although TSP1 and the peptide conjugates inhibited the growth of both cell types. Apoptosis did not depend on activation of latent transforming growth factor beta because peptides lacking the activating sequence RFK were active. Apoptosis was not sensitive to inhibitors of ceramide generation but was inhibited by the phosphatase inhibitor vanadate. Induction of DNA fragmentation by the peptides was decreased when endothelial cell cultures reached confluence. Growth of the cells on a fibronectin substrate also suppressed induction of apoptosis by TSP1 or the peptides. Differential sensitivities to kinase inhibitors suggest that apoptosis and inhibition of proliferation are mediated by distinct signal transduction pathways. These results demonstrate that induction of apoptosis by the TSP1 analogues is not a general cytotoxic effect and is conditional on a lack of strong survival-promoting signals, such as those provided by a fibronectin matrix. The antitumor activity of TSP1 may therefore result from an increased sensitivity to apoptosis in endothelial cells adjacent to a provisional matrix during formation of vascular beds in tumors expressing TSP1.