A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5±3.6, 79.17±4.5 and 13.25±4.7%, respectively. The fertilization rates of this sperm were 67.06±15.1, 76.20±10.0 and 44.93±22.6%, while the hatching rates of eggs fertilized with this sperm were 37:40 8:3, 48:18 25:7 and 23:35±0.08%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen–thawed sperm.