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OBJECTIVE: To examine simultaneously the levels of hydrogen peroxide (H₂O₂) and superoxide (O₂^−•) using chemiluminescence and flow cytometry. DESIGN: Prospective laboratory study. SETTING: Reproductive research lab in a tertiary hospital. PATIENT(S): Semen samples from 18 healthy male volunteers. INTERVENTION(S): Sperm preparation and measurement of reactive oxygen species (ROS) by chemiluminescence using luminol and lucigenin before and after H₂O₂ exposure and by flow cytometry using dichlorofluorescin diacetate (DCFH-DA) for H₂O₂ and dihydroethidium (DHE) for O₂^−•. MAIN OUTCOME MEASURE(S): Sperm count, motility, viability, and ROS levels. RESULT(S): Immature sperm fractions showed significantly higher levels of ROS measured by either luminol or lucigenin compared with the neat and mature fraction. ROS levels were detectable by flow cytometry in chemiluminescence …