Objective. The aim of the present study was to analyze α2Macroglobulin/MMP (α2M/MMP) complex formation and to investigate whether MMP activity in α2M/MMP complexes in serum can be used as a disease marker in rheumatoid arthritis (RA). Methods. High and low molecular weight (H/LMW) substrates and inhibitors and size exclusion were used to analyze α2M/MMP complex formation. LMW fluorogenic substrates were used to quantify the level of MMPs in α2M/MMP complexes in the serum of RA patients and healthy controls. Results. Active MMPs were fully inhibited by LMW inhibitor BB94 in the presence of α2M, whereas no inhibition was achieved by HMW inhibitor TIMP-1. Size exclusion analysis showed α2M/MMP complex formation in buffer and in normal plasma spiked with activated MMPs, whichFrom Subject Received Size Categories Beheer-TNO-SharePoint NDIA Weekly Insider 8:00 18 KB indicated α2M/MMP complex formation in the systemic circulation. MMP activity in α2M/MMP complexes in the serum of RA patients was significantly higher than in the serum of healthy controls (P<0.001). MMP activity levels in the serum of RA patients were correlated with ESR (r = 0.72, P<0.001). Conclusion. In the systemic circulation of RA patients, active MMPs form complexes with α2M and can be detected using LMW fluorogenic substrates. MMP activity measurements in serum allow discrimination between RA patients and healthy controls and provide a new tool for the assessment of the disease process in RA.