Early detection of osteoarthritis in horses represents a challenge for equine practitioners. Several biological markers have been implicated in the pathological processes involved in articular cartilage destruction. To further document cartilage matrix proteases production, synovial fluid was collected from 14 horses (90 joints) before they were subjected to euthanasia. Growth macroscopic examination of the joints gave information on cartilage alterations. Samples were analyzed for matrix metalloproteinase (MMPs) activities by gelatin zymography and tumor necrosis factor alpha (TNF-alpha) cytotoxicity using L929 cells. Significant increase of MMP-9 monomer and dimer were found in synovial fluids of joints with severe cartilage alterations. On the contrary, the activity of TNF-alpha was not correlated to the degree of joint damage. The levels of MMP-9 monomer and dimer in the synovial fluid could reflect cartilage alteration in arthritis in the horse.