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BACKGROUND: The aim of this study was to examine the role of apoptosis and reactive oxygen species (ROS) in inducing DNA damage in ejaculated spermatozoa. METHODS: We examined ejaculated spermatozoa from 31 patients examined for infertility and 19 healthy donors for apoptosis, production of ROS and DNA damage using annexin V binding, chemiluminescence assay and sperm chromatin structure assay. RESULTS: The percentage of spermatozoa that underwent apoptosis in the whole ejaculate and mature fraction was higher in the patients than in the donors (P < 0.001 and P = 0.009, respectively). Levels of ROS in the whole ejaculate and immature fraction were higher in the patients than in the donors (P = 0.002 and P = 0.009). Apoptosis was significantly correlated with ROS within patients in the whole ejaculate [r (95% confidence interval) = 0.53 (0.19–0.86)] and in the mature [0.71 (0.39–1 …