Context: Defective sperm function is the largest defined cause of human infertility; however, the etiology of this condition is poorly understood. Although oxidative stress is acknowledged as a key contributor to this pathology, there are also data indicating that defective human spermatozoa contain abnormally high amounts of cis-unsaturated fatty acids. This study investigated whether a causative relationship exists between these two attributes of impaired semen quality. Objective: The objective of this study was to determine whether polyunsaturated fatty acids can induce oxidative stress in human spermatozoa. Method: Dihydroethidium and SYTOX Green were used in conjunction with flow cytometry and HPLC to investigate reactive oxygen species (ROS) generation by human spermatozoa after fatty acid exposure. Results: Arachidonic acid (AA) induced a time- and dose-dependent increase in ROS generation by human spermatozoa that led to the promotion of peroxidative damage and a loss of sperm motility. This effect could not be blocked with inhibitors of the cyclooxygenase or lipoxygenase pathways of AA metabolism, rotenone, protein kinase C antagonists, or known inhibitors of plasma membrane redox systems. However, ROS generation could be triggered with other cis-unsaturated fatty acids including linoleic and docosahexaenoic acids. Saturated fatty acids, methyl esters of unsaturated fatty acids, or other amphiphiles were all ineffective. However in a cell-free system, AA could trigger a redox signal via mechanisms that were profoundly disrupted by diphenylene iodonium, a flavoprotein inhibitor. Conclusions: The presence of excess unsaturated fatty acids in defective human spermatozoa may precipitate the oxidative stress encountered in male infertility.