A 1 h, one-step sandwich enzyme immunoassay was set up with a pair of monoclonal antibodies prepared against bovine tissue inhibitor of metalloproteinases (TIMP). TIMP in samples was allowed to react simultaneously with both solid-phase antibody and peroxidase-labeled Fab' of another antibody. Minimum sensitivity was 1 pg/well for bovine TIMP and 1.5 pg/well for human TIMP. The TIMP level in the sera of 38 rheumatoid arthritis (RA) patients (241 +/- 53 ng/ml, mean +/- SD) was significantly higher (P less than 0.001) than the level in the sera of 81 healthy subjects (175 +/- 39). A similar significant difference (P less than 0.001) was observed between the TIMP level in platelet-poor plasma of normal subjects (64 +/- 10) and that of RA patients (84 +/- 23), suggesting that the increase in TIMP seen in RA sera can not be ascribed merely to an increase in platelet-derived TIMP.