The process of angiogenesis is important in both normal and pathologic physiology. However, the mechanisms whereby factors such as basic fibroblast growth factor promote the formation of new blood vessels are not known. In the present study, we demonstrate that exogenously added plasminogen activator inhibitor-1 (PAI-1) at therapeutic concentrations is a potent inhibitor of basic fibroblast growth factor-induced angiogenesis in the chicken chorioallantoic membrane. By using specific PAI-1 mutants with either their vitronectin binding or proteinase inhibitor activities ablated, we show that the inhibition of angiogenesis appears to occur via two distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1's anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.