The effect of some antioxidants on acrosin amidase activity of chilled stallion spermatozoa was investigated in this study. Nine ejaculates were collected from six Arabian horses. A comparison between four antioxidants namely, sodium pyruvate (0.5 mg/ml), sodium thiosulfate (STS, 1.0 mg/ml), bovine serum albumin (BSA, 5.0 mg/ ml), zinc chloride (0.15 mg/ml) and a mixture of them was studied in a chemically-defined stallion semen extender (Tris-egg yolk) at 5°C. The comparison was based on sperm viability, acrosin amidase activity and changes in the levels of extracellular alanine aminotransferase (ALT). Results of the present experiments revealed that sodium pyruvate and the mixture of antioxidants were most effective for improving viability and acro- sin amidase activity of stallion spermatozoa. Low values of ALT in the extracellular medium were coincided with high values of acrosin amidase activity of equine stallion spermatozoa during storage at 5°C.