The human epididymis provides an optimal environment for the storage and maturation of spermatozoa. However, the ability of the epididymis to protect spermatozoa from oxidative attack whilst stored at this site, through the local actions of antioxidants, has not thus far been well studied. This study assessed the contribution of the epididymis to seminal plasma antioxidant activity, by comparing the semen of normozoospermic and vasectomized men. Total seminal plasma antioxidant activity was measured, as were concentrations of urate, ascorbate and thiols, antioxidants that are abundant in human semen. Thiobarbituric acid reactive species (TBARS) were measured to indicate lipid peroxidation. Total antioxidant activity and thiol content were significantly lower (P < 0.05) in the plasma from vasectomized men compared with that of normozoospermic donors. Ascorbate and urate were found at similar concentrations in the plasma of both groups. The concentration of TBARS was significantly higher (P < 0.001) in the semen from vasectomized individuals compared with the normozoospermic group. The results indicate that the epididymis contributes to the antioxidant capacity of seminal plasma and possesses region-specific antioxidant activity, which may potentially protect spermatozoa from oxidative attack during storage at this site.