In order to establish a reliable and reproducible method for cryopreservation of medaka spermatozoa, we tested several procedures. The vapor phase of liquid nitrogen (LN), the liquid phase of LN, and dry ice were used for freezing, and dimethyl sulfoxide and N, N-dimethylformamide (DMF) were used as cryoprotectants. The best results were obtained using the following method. Medaka spermatozoa were collected in a plastic tube containing 50 μl of fetal bovine serum supplemented with 10% DMF by squeezing an isolated testis. The sperm suspension was frozen by holding the tube for 10 or 20 min in the vapor phase of LN at a depth of 9 or 10 cm from the top of a container. The frozen sample was immersed and stored in LN. After more than one week of storage, the sample in the tube was rapidly thawed by being incubated in a waterbath for 0.5–1 min at 30°C, and then immediately diluted with 2 volumes of Iwamatsu's solution. Fertilization tests using fresh unfertilized eggs showed that the stored spermatozoa could fertilize 96–100% of the eggs. The hatchability of the fertilized eggs was 84-100%. Thus, this study provides, for the first time, a practical method for preserving medaka spermatozoa.