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The purpose of this study was to investigate whether synovial fluid levels of matrix metalloprotease-3 (MMP-3) and tissue inhibitor of metalloprotease-1 (TIMP-1) are specifically elevated in canine rheumatoid arthritis (CRA) compared to osteoarthritic joint disorders and if these markers are correlated with a specific pattern of cytokine mRNA expression. Synovial fluid samples of 17 dogs with CRA were analysed for MMP-3 and TIMP-1 by two canine sandwich ELISA (enzyme-linked immunosorbent assay) systems. The synovial mRNA content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma) was determined by RT-PCR (reverse transcription-polymerase chain reaction). Dogs with osteoarthritis (n = 50) caused by anterior cruciate ligament rupture (ACLR) were used as controls. A significant rise of MMP-3 was found in the synovial fluid of joints with CRA that could not be balanced by sufficient amounts of TIMP-1. The 30-fold surplus of MMP-3 over TIMP-1 was strongly correlated with the synovial mRNA content of IL-1, IL-12 and TGF-beta. Our results point to the potential use of the synovial levels of MMP-3 as a marker for RA in dogs.