STUDY HYPOTHESIS: Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? STUDY FINDING: Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. WHAT IS KNOWN ALREADY: MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: MOMP was associated with ΔΨm dissipation (P < 0.05), increased ROS production (P < 0.05) and decreased mean sperm velocity (P < 0.05), but it was not associated with DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study in male infertility. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: The study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT (F.T.). The authors declare no conflict of interest.