We describe a library of two-chain molecular complementation mutants o
f staphylococcal alpha-hemolysin that features a combinatorial cassett
e encoding thousands of protease recognition sites in the central pore
-forming domain. The cassette is flanked by a peptide extension that i
nactivates the protein. We screened the library to identify alpha-hemo
lysins that are highly susceptible to activation by cathepsin B, a pro
tease that is secreted by certain metastatic tumor cells. Toxins obtai
ned by this procedure should be useful for the permeabilization of mal
ignant cells thereby leading directly to cell death or permitting dest
ruction of the cells with drugs that are normally membrane impermeant.