Context: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved. Objective: The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function. Method: DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells. Results: HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage. Conclusions: These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.