Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 µg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the "C-Ruch" computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P