BACKGROUND: Reactive oxygen species (ROS)-induced damage of membrane phospholipids and DNA in human spermatozoa has been implicated in the pathogenesis of male infertility. In this study, variations in ROS production, DNA structure (as measured by the sperm chromatin structure assay) and lipid composition, were studied in human spermatozoa at different stages of maturation. METHODS: Sperm subsets were isolated by discontinuous density gradient centrifugation of semen samples obtained from healthy donors and from infertility patients. RESULTS: DNA damage and ROS production were highest in immature spermatozoa with cytoplasmic retention and abnormal head morphology, and lowest in mature spermatozoa. Docosahexaenoic acid and sterol content were highest in immature germ cells and immature spermatozoa, and lowest in mature spermatozoa. The relative proportion of ROS-producing immature spermatozoa in the sample was directly correlated with DNA damage in mature spermatozoa, and inversely correlated with the recovery of motile spermatozoa. There was no correlation between DNA damage and sperm morphology in mature spermatozoa. CONCLUSIONS: The high levels of ROS production and DNA damage observed in immature spermatozoa may be indicative of derangements in the regulation of spermiogenesis. DNA damage in mature spermatozoa may be the result of oxidative damage by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis.