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The objective of this study was to investigate the concentrations of interleukin-1 receptor antagonist (IL-1Ra) and interleukin-1ß (IL-1ß) in synovial fluid (SF) and serum (SE) of horses with different grades of osteoarthritis (OA) and septic arthritis. Based on SF analysis, radiographic, and arthroscopic scores, 40 horses were classified into three groups as follows: mild OA, advanced OA, and septic arthritis. Horses without orthopedic problems served as a control group. Equine-specific antibody enzyme-linked immunosorbent assays were used to determine the concentration of IL-1Ra and the catabolic cytokine IL-1ß in SF and SE. Results were further compared with levels of the previously described biomarkers C-telopeptide fragments of type II collagen (CTX-II) and myeloperoxidase. In the present study, the SF of healthy joints, those with nonseptic OA, and those with septic arthritis contained significantly different levels of IL-1Ra. Serum concentrations of IL-1Ra were only significantly elevated in horses with septic arthritis when compared with the control group. Different levels of IL-1ß were detected in SE of control horses compared with those with various degrees of joint disease. Synovial fluid concentrations of IL-1ß were only moderately elevated in the groups of horses with joint disease. In addition to lameness examination and standard diagnostic procedures, the determination of IL-1Ra concentration in SF, in combination with further biomarkers, might be useful to assess the extent of intrasynovial inflammation.