To determine whether flow sorting increased the susceptibility of spermatozoa to reactive oxygen species (ROS), ram semen was either diluted with Tris medium (100 × 10⁶ spermatozoa mL^–1; D) or highly diluted (10⁶ spermatozoa mL^–1) before being centrifuged (DC) at 750g for 7.5 min at 21°C or flow-sorted (S) before cryopreservation. Thawed spermatozoa were resuspended in graded concentrations of hydrogen peroxide to induce oxidative stress. In Experiment 1, following exposure to 30 or 45 μM hydrogen peroxide (H₂O₂), the total motility (%) of DC (41.0 ± 7.3 or 25.7 ± 6.7, respectively) and S spermatozoa (33.8 ± 6.3 or 20.1 ± 6.3, respectively) was lower (P < 0.001) than that of D spermatozoa (58.7 ± 5.6 or 44.5 ± 6.7, respectively). In Experiment 2, supplementation of samples containing H₂O₂ with catalase (150 IU mL^–1) or seminal plasma proteins (4 mg protein per 10⁸ spermatozoa) negated oxidative stress, resulting in comparable values to samples receiving no H₂O₂in terms of the proportion of spermatozoa with stable plasmalemma (as determined using merocyanine-540 and Yo-Pro-1) in the D and S groups, the proportion of viable, acrosome-intact spermatozoa (as determined by fluorescein isothiocyanate and propidium iodide staining) in the D group and the motility of control (undiluted) and S spermatozoa. Neither H₂O₂ nor sperm type (i.e. D, DC or S) had any effect on intracellular concentrations of ROS. These results show that flow sorting increases the susceptibility of spermatozoa to ROS, but the inclusion of anti-oxidants or seminal plasma as part of the sorting protocol improves resistance to oxidative stress.