The objective of this study was to compare measurements of reactive oxygen species (ROS) generation from human spermatozoa in vitro using the luminol and lucigenin chemiluminescent probes. Luminol reacts with a variety of reactive oxygen species (H2O2, O2-, OH) and allows both intra- and extracellular ROS to be measured. Lucigenin, however, yields a chemiluminescence that is more specific for superoxide anions released extracellularly. Therefore, measurements made with both probes on the same samples should allow the intra- and extracellular components of ROS generation to be identified. Sperm samples from 47 men were divided into two equal aliquots, then processed by centrifugation and swim-up. Following further division into aliquots and the addition of the two chemiluminescent probes, Phorbol 12-myristate 13-acetate was added to trigger ROS release. Forty three percent of the sperm samples generated detectable levels of ROS. In the centrifuged preparations luminol produced a significantly higher peak luminescence than lucigenin. However, the sperm prepared by swim-up showed no significant differences in peak luminescence between luminol and lucigenin. The higher level of ROS generation produced by centrifugation may be due to membrane disruption or possibly the use of unfractionated cell suspensions. Extracellular ROS generation is more clinically important because surrounding healthy spermatozoa may be damaged. Therefore the lucigenin probe may be a more useful diagnostic tool than luminol for identifying sperm at risk of peroxidative damage after swim up preparation. The patients identified in this way may benefit from the addition of ROS scavengers to the culture medium in order to protect healthy sperm from collateral damage.