Objectives - To determine concentrations of matrix metalloproteinase (MMP)-2 and -9 in synovial fluid; and mRNA expression of MMP-1, -13, and -3; interleukin[IL]-1α and β; and tumor necrosis factor(TNF)-α in synovial membrane and articular cartilage from horses with naturally occurring joint disease. Sample Population - Synovial fluid (n = 76), synovial membrane (59), and articular cartilage (45) from 5 clinically normal horses and 55 horses with joint disease categorized as traumatic (acute [AT] or chronic [CT]), osteochondritis dissecans (OCD), or septic (S). Procedure - Synovial fluid gelatinase concentrations were analyzed, using zymography. Synovial membrane and articular cartilage mRNA expression for MMP-1, -3, and -13, IL-1α and β, TNF-α, type-II collagen, and aggrecan were analyzed, using quantitative reverse transcriptase-polymerase chain reaction. Results - Synovial fluid pro-MMP-2 concentration was significantly higher in diseased joints than normal joints. Septic joints had significantly higher concentrations of pro and active MMP-9. Stromelysin-1 was expressed in ≥ 80% of synovial membrane and articular cartilage samples and was strongly influenced by age. Collagenases were rarely expressed, with MMP-13 expressed only in diseased joints. Interleukin-1β expression was significantly higher in all OCD samples and was influenced by age. Tumor necrosis factor-α expression was significantly higher in cartilage from joints with AT and OCD. There was no correlation between MMP or cytokines and type-II collagen or aggrecan expression. Conclusions and Clinical Relevance - Matrix metalloproteinase-2 and -3 are abundant in naturally occurring joint disease and normal joints, Interleukin-1β and TNF-α may be important in the pathogenesis of OCD. Age affects MMP and IL-1β concentrations.