We obtained 69,168 naturally spawned eggs of the ocellate puffer from 11 sampling stations in the spawning ground off Anori, Mie Prefecture, using a skidnet on the 15th and 23rd April, 1993. Naturally spawned eggs were not distributed uniformly. The number of days reqired until hatching varied among the stations and was estimated to range from 4 to 10 days and a considerable number of parental fish were estimated to be involved in spawning. Fry from 4 stations were raised to an average standard length of 8.5~11.9 cm and then PGM isozyme in muscle and IDH isozyme in liver of fry were analyzed by horizontal starch-gel electrophoresis. The PGM-c gene frequencies of St. 4 and St. 10 in the eastern region were 0.806 and 0.816, respectively, whereas in the western region, St. 6 and St. 11 were 0.621 and 0.608, respectively. The IDH-N gene frequency of St. 4 was 0.707, in contrast that of St. 11 was 0.539. These results imply that two spawning groups, one in each of the eastern and western areas of this spawning ground exsisted. However, it is necessary to examine whether these spawning groups correspond to stocks. Moreover the number of parent fish spawned in the spawning ground were estimated to be at least 61 to 233 individuals from the frequencies of the rare genotypes and genes. These estimations obtained from isozyme analysis are supported by data on the distribution and hatching of naturally spawned eggs.