The withdrawal time of erythromycin in cultured olive flounder was investigated to ensure the food safety of the fish treated with erythromycin. The oral administration of erythromycin was carried out using the experimental diet containing erythromycin (200 mg/kg) daily dose of 40 mg/kg body weight. The 45 day experimental period was broken into 7 days of habituation, 8 days of medication and 30 days of additional feeding without antibiotics. The erythromycin concentration in the flounder muscle had been increased gradually with medication. After 5 days of medication, the concentration increased to its maximum level of 6.05 mg/kg. After discontinuing the antibiotic, the erythromycin concentration decreased drastically and day 9 was below 0.1 mg/kg. The erythromycin concentration had slowly declined from the 6th to the 20th day after medication and disappeared completely after 25 days. From these results, the time needed to reduce the erythromycin level to the 0.2 mg/kg limit adopted by the ED and Japan was suspected to be 4-6 days. Therefore, a reasonable withdrawal time following ED and Japanese regulatory guidelines for erythromycin in the cultured flounder could be estimated to be 10 days.