Tumor cells traverse basement membranes (BM) during the stages of the metastatic process. Penetration of the BM may involve proteolysis by enzymes directly or indirectly associated with tumor cells. This study evaluated the role of the serine proteases urokinase (plasminogen activator), plasmin, and another regulatory protease, α-thrombin, in the degradation of the BM. Homogeneously pure enzyme preparations were incubated with isolated components of BM and with whole human amnion BM. The BM components consisted of acid-extracted type IV collagen, pepsin fragments of collagen type IV, laminin, and fibronectin. Collagen type V (αAαB) associated with the peri-BM zone was also studied. The purity of the enzymes was verified by gel electrophoresis and inhibitor studies. Digestion of the BM components was performed at 25° using matched activity for the different enzymes. Urokinase failed to significantly degrade fibronectin or any of the other BM components. Under the same 25° (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or αAαB (type V) collagen. α-Thrombin selectively degraded only the m.w. 400,000 chain of laminin, whereas plasmin degraded both of the laminin chains. Digestion of laminin by the serine proteases was time and concentration dependent, as verified by a new degradation assay using [¹⁴C]laminin. A variety of normal and neoplastic cells were tested for the presence of laminin-degrading proteases. Macrophages, polymorphonuclear leukocytes, and metastatic tumor cells contained a significant laminin-degrading activity. The activity was enhanced by the addition of plasminogen. Type V collagen was cleaved by thrombin and plasmin at 35° but not at temperatures below 33°. Following treatment of whole-amnion BM with any of these enzymes, electron microscopy demonstrated preservation of the lamina densa. Immunohistology studies indicated that laminin, but not type IV collagen, was removed from the whole BM by plasmin treatment. The results suggest that these BM components are poor substrates for plasminogen activators and that plasmin alone is not sufficient to completely degrade the whole BM. Plasmin, generated through the action of plasminogen activator, may play a significant role in the degradation of noncollagenous components of the BM.