The objectives of this study were to determine if human ejaculated sperm exhibit active caspases and if caspase-dependent apoptosis markers are identifiable. Sperm from fertile donors and infertile patients were examined after gradient separation into leukocyte-free fractions of high and low motility. Sperm were evaluated for motion parameters, morphology, caspase activation, and apoptosis markers including phosphatidylserine (PS) translocation (annexin V binding) and DNA fragmentation (TUNEL). Active caspase-3 was detected by immunofluorescent microscopy in a small proportion of sperm in situ, in fractions of high and low motility sperm of patients and donors, but low motility fractions had significantly higher numbers of positive sperm. Immunoblot analysis detected inactive procaspase-3 (32 kDa) in all fractions of low sperm motility from patients and donors, while active caspase-3 (17 kDa) was only detected by immunoblotting in a limited number of low motility fractions from patients and in even fewer fractions from donors. Caspase enzymatic activity, as measured using the fluorogenic substrate DEVD-afc, was higher in patients than in donors in both low and high motility fractions. Annexin V staining and DNA fragmentation were detected in a proportion of sperm, with a higher frequency in the low motility fractions. A significant positive correlation between in-situ active caspase-3 in the sperm midpiece and DNA fragmentation was observed in the low motility fractions of patients, suggesting that caspase-dependent apoptotic mechanisms could originate in the cytoplasmic droplet or within mitochondria and function in the nucleus. These data suggest that in some ejaculated sperm populations, caspases are present and may function to increase PS translocation and DNA fragmentation.