Highly purified alpha-thrombin, plasmin and urokinase were incubated with laminin, type IV collagen and type V collagen. At 25 degrees C (1:100 enzyme to substrate ratio on a weight/weight basis) alpha-thrombin selectively degraded the beta chain of the native laminin, whereas plasmin degraded both the alpha and beta chains. The specific limited cleavage fragments of laminin produced by alpha-thrombin and plasmin retained the ability to mediate binding of epithelial cells to type IV collagen. These serine proteases failed to degrade native type IV or V collagen under identical experimental conditions. At 35 degrees C type V collagen, but not type IV collagen, was partially cleaved by both alpha-thrombin and plasmin. Urokinase failed to degrade any of the substrates. Specific cleavage products are used for structural and immunological studies.