Mouse monoclonal antibodies against recombinant human fibroblast procollagenase and prostromelysin have been generated and characterized. The epitope-containing domains for the antibodies have been assigned based on their immunoreactivities against recombinant proenzymes, mature enzymes, truncated collagenases, proteolytic fragments of stromelysin, and chimeric molecules constructed from different domains of the two enzymes. These antibodies can be divided into four groups: (1) antibodies that recognize the truncated 19-kDa NH2-terminal collagenase, (2) antibodies that recognize the C-terminal domain of collagenase and stromelysin, (3) antibodies that recognize a 31-kDa NH2-terminal collagenase fragment, and (4) antibodies that recognize the 19-kDa NH2-fragment of stromelysin. The prostromelysin-specific antibody 11N13 is of particular interest; it neutralizes stromelysin activity in a stromelysin peptide substrate assay, with an IC50 value of 75 nM. MAb 11N13 may be useful for in vivo and in vitro studies to validate the roles of stromelysin in tumor cell invasion, metastasis, and connective tissue disorders.