Matrix metalloproteinases (MMPs) may be important in the destruction of cartilage seen in equine osteoarthritis and may be detectable in synovial fluid. Synovial fluids were obtained from normal equine joints and from joints of horses with aseptic and septic joint diseases. The total MMP gelatinase enzyme activities were measured by gelatin zymography and image analysis of the gels. The bioactivity of gelatinase in synovial fluid was determined by a gelatin degradation ELISA. Potential MMP-2 & MMP-9 monomer enzyme activities were significantly elevated in both septic and aseptic joint disease synovial fluids in comparison to fluids from normal joints. The dimer form of MMP-9 enzyme activity was significantly elevated in fluids from septic joint disease cases in comparison to fluids form normal joints, but not fluids from horses with aseptic joint diseases. MMP-9 monomer and dimer levels in synovial fluids correlated with the synovial fluid white blood cell count. Using the gelatin degradation ELISA to measure net active gelatinases, significant increases in gelatinase bioactivities were seen in synovial fluids from both aseptic and septic joint disease cases. The enzymes in equine joint diseases were present in a bioactive form, in that they were present in the activated form and present in excess of inhibitors, and could therefore be important in the degradation of articular cartilage in joint disease.