Thrombospondin-1 (TSP-1) is a potent antiangiogenic factor that has been shown to inhibit tumor growth by preventing endothelial cells from responding to a wide variety of angiogenic stimulators. We have demonstrated previously that D-12 primary tumors (human melanoma xenografts) suppress the growth of their spontaneous pulmonary micrometastases by secreting TSP-1 into the blood circulation. The same tumor model was used in the present work to study antitumor effects of combined radiation therapy and antiangiogenic treatment with TSP-1. Curative radiation treatment of D-12 primary tumors resulted in rapid growth of previously dormant micrometastases. Growth of dormant micrometastases could be prevented by treating the host mice with exogenous TSP-1 after the radiation treatment. Treatment with exogenous TSP-1 after subcurative radiation treatment reduced the growth rate of recurrent primary tumors in addition to suppressing metastatic growth. TSP-1 suppressed tumor growth at both primary and metastatic sites by inducing apoptosis in tumor-associated microvascular endothelial cells. Treatment with exogenous TSP-1 before radiation treatment enhanced the antitumor effect of the radiation treatment. The radiopotentiation by TSP-1 involved at least two distinctly different mechanisms, i.e., TSP-1 reduced the fraction of radiobiologically hypoxic parenchymal tumor cells and increased the radiation sensitivity of the tumor microvasculature by promoting radiation-induced endothelial cell apoptosis. In conclusion, the present preclinical study showed that TSP-1 has antiangiogenic, antimetastatic, and radiopotentiating properties that merit additional investigation in clinical studies.