Mouse monoclonal antibodies directed against cathepsin L and procathepsin L have been generated. Mice were immunized with human procathepsin L purified from the cell culture medium of human nonsmall cell lung cancer cell line EPLC 32 Ml. More than 400 hybridoma clones were screened by ELISA or Western blot and 50 were found to secrete antibodies which reacted with cathepsin L or its precursor. Twenty-six clones were selected for further characterization of the antibodies according to their reactivity in ELISA, Western blot, and immunocytochemistry against the mature enzyme or its latent precursor of man, rat, or mouse. Only those antibodies are described here, which do not cross-react with the closely related cathepsins B and S or their latent proenzymes.