OBJECTIVE: To investigate the ability of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), complement 5a (C5a), and nerve growth factor (NGF) to stimulate human spermatozoal reactive oxygen species generation in fertile and infertile patients. DESIGN: Prospective, controlled study measuring human spermatozoal reactive oxygen species generation after addition of f-MLP, C5a, or NGF. SETTING: A large health maintenance organization. PATIENTS, PARTICIPANTS: The fertile group consisted of 14 men with established fertility and normal bulk semen parameters. The infertile group was comprised of 8 men who were infertile after > 18 months of unprotected sexual intercourse. INTERVENTIONS: The sperm samples were subjected to four test conditions: f-MLP stimulation, C5a stimulation, NGF stimulation, and no stimulation (control). MAIN OUTCOME MEASURE: Reactive oxygen generation was measured over a 15-minute period using the method of chemiluminescence. RESULTS: In both the fertile and infertile groups, reactive oxygen species generation was significantly enhanced by f-MLP, C5a, and NGF compared with controls. No significant difference in f-MLP- and C5a-stimulated reactive oxygen production was demonstrated between the infertile and fertile groups; however, there was a significant difference in reactive oxygen generation between infertile and fertile subjects when stimulated with NGF. CONCLUSIONS: The current study represents the first report of f-MLP-, C5a-, and NGF-stimulated reactive oxygen species generation by human spermatozoa. Nerve growth factor enhanced reactive oxygen species production to a greater extent in infertile subjects compared with fertile subjects. This points to a possible NGF-mediated biochemical defect in the sperm of infertile patients.