A one-step sandwich enzyme immunoassay (EIA) for human matrix metalloproteinase 2 (MMP-2, 72-kDa gelatinase/type IV collagenase, EC 3.4.24.24) was established with a pair of monoclonal antibodies prepared against the precursor form of MMP-2 (proMMP-2) purified from the conditioned medium of human skin fibroblasts or against a synthetic peptide corresponding to the N-terminal domain of proMMP-2. ProMMP-2 in samples was allowed to simultaneously react with both solid-phase and peroxidase-labeled antibodies. Sensitivity of this EIA system was 2.4 pg/assay (0.24 microgram/l) and linearity was obtained between 10 and 5,000 pg/assay (1.0-500 micrograms/l). The EIA system recognized both the free form of proMMP-2 and its complex form with TIMP-2 with the same degree of immunoreactivity. ProMMP-2 levels in human sera from patients in various disease states were analyzed. In sera from patients with hyperthyroidism (12), primary biliary cirrhosis (8) and hepatocellular carcinoma (11), 749 +/- 166, 716 +/- 135 and 686 +/- 236 micrograms/l of proMMP-2 were detected, respectively and these were significantly higher than that observed in 213 normal human sera (570 +/- 118 micrograms/l). In contrast, the levels in sera from 33 patients with osteoarthritis (449 +/- 72 micrograms/l), 45 with rheumatoid arthritis (408 +/- 139 micrograms/l), 13 with stomach cancer (427 +/- 103 micrograms/l) and 10 with pancreatic cancer (422 +/- 130 micrograms/l) were significantly lower than that found in normal sera. Immunoblot and gel filtration analyses showed that human sera contain several MMP-2 species in addition to proMMP-2 which exist in a complex form with TIMP-2.