We studied the state of alpha 2-macroglobulin (alpha 2M), an important inhibitor of cartilage-degrading proteinases, in relation to activation of neutrophils in 82 patients with several types of arthritis, including 52 with rheumatoid arthritis and 11 with osteoarthritis. Levels of total inactive alpha 2M (i alpha 2M), which comprises alpha 2M complexed to proteinases and alpha 2M inactivated by oxidation or hydrolysis, were measured with a monoclonal antibody specific for i alpha 2M. In addition, levels of alpha 2M complexed to proteinases were quantitated with specific assays. Neutrophil activation was assessed by measuring elastase-alpha 1-antitrypsin complexes and lactoferrin. In 83% of the 82 patients tested, the synovial fluid (SF) to plasma ratio of i alpha 2M exceeded 1, indicating an intraarticular generation. Levels of i alpha 2M significantly correlated with neutrophil numbers (P less than 0.0005) and with levels of elastase-alpha 1-antitrypsin complexes and of lactoferrin (P less than 0.00001 for both). Moreover, part of i alpha 2M consisted of alpha 2M complexed to elastase-like and chymotrypsin-like proteinases, presumably, neutrophil elastase and cathepsin G, respectively. However, the amount of i alpha 2M was approximately 10-fold larger than the amount complexed to these proteinases. In vitro inactivation of alpha 2M by activated neutrophils was only partly inhibitable by eglin C, a specific inhibitor of both elastase and cathepsin G. Release of reactive oxygen species was presumably responsible for the additional inactivation of alpha 2M, because eglin C completely abolished the inactivation of alpha 2M by cell-free supernatant of activated neutrophils. Thus, our results suggest a predominant role of neutrophils in the inactivation of alpha 2M in the SF of patients with inflammatory joint diseases. However, this inactivation could be explained only in part by the release of neutrophilic proteinases. We propose that the inactivation of alpha 2M in SF was due to the concerted action of both reactive oxygen species and lysosomal proteinases.