Cathepsin B activity, including that of a plasma membrane-associated cathepsin B, has been linked to tumor malignancy. As cathepsin B at the tumor cell surface has been hypothesized to play a role in the focal degradation of basement membrane during the metastatic cascade, we have examined the ability of human tumor cathepsin B to degrade laminin, an adhesive glycoprotein found exclusively in the basement membrane. We report that at pH 6.5 and 7.0 tumor cathepsin B degraded by specific, limited proteolysis both subunits of native laminin. The disappearance of both subunits and the appearance of lower Mr protein bands could be observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Accumulation of degradation products was also observed using gel filtration chromatography and a fluorescamine assay. The proteolysis of laminin by tumor cathepsin B could be inhibited by an active site titrant for cysteine proteinases or stefin B, an endogenous low-Mr cysteine proteinase inhibitor.