Physical, biochemical and cytological characteristics of the digital flexor tendon sheath synovial fluid were studied in nine clinically normal horses. It is a viscous, pale yellow, clear fluid, without flocculent material and does not clot at room temperature. Its hyaluronic acid mean concentration was 60.20mg/dL, glucose levels were similar to the plasmatic levels, and its protein concentration was up to 1.74g/dL, with mean albumin:globulin ratio of 0.94. Nucleated cells mean number was 313 cells/µL, with predominating large mononuclear cells and lymphocytes. There was a significant correlation (r= -0.649, P<0.01) between increasing hyaluronic acid concentration and decreasing lymphocyte counts. Equine digital tendon sheath synovial fluids can be easily evaluated, being feasible in clinical and surgical routine, and the information may be useful to the diagnosis, treatment and prognosis of animals with tendon sheath associated lesions.