The proteasome is a recently identified intracellular protease whose catalytic active site is a threonine residue and has been shown to play key roles in a variety of important intracellular events, including cell cycle progression, the antigen-presenting pathway, and apoptosis. However, its biological significance in multicellular organisms is still largely unknown because of lack of experimental systems for its study. Here we verified potential involvement of the proteasome in angiogenesis using lactacystin, a specific proteasome inhibitor. Lactacystin treatment resulted in almost complete prevention of in vivo neovascularization in the developing chick embryo chorioallantoic membrane. It also inhibited vascular endothelial tube formation on Matrigel, a model for in vitro angiogenesis, in a concentration-dependent fashion. Moreover, it prevented production of plasminogen activator, an important protease responsible for induction of angiogenesis, by endothelial cells, which correlated well with its suppression of intracellular proteasome activity. Our studies suggest that the proteasome operates in the process of angiogenesis, a phenomenon essential in important physiological and pathological settings.