Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10 2 cell/ml sperm concentration and a contact time of 2min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60min at 25 o C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5ml microtubes and frozen using a cooling rate between -3.5 and -20 o C/min to various transition temperatures (0, -30, -60, -90 and -120 o C), followed by transfer and storage in liquid nitrogen (-196 o C). The microtubes were thawed from +45 to +145 o C/min. Spermatozoa, cooled to -90 o C at a cooling rate of -12 or -15 o C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.