Growth and amino acid oxidation studies were conducted to estimate methionine requirement of juvenile Japanese flounder, Paralichthys olivaceus, by using the purified diets containing 500 g kg–1 crude protein from casein, gelatine and crystalline amino acids (CAA). Diets with six graded levels of methionine (5.3, 8.3, 11.3, 14.3, 17.3 and 20.3 g kg–1 diet) were fed to triplicate groups of the juvenile (initial weight 2.8 ± 0.05 g) twice a day for 40 days. To prevent leaching losses, CAA were precoated using carboxymethylcellulose (CMC), and further diets were bound by CMC and κ-carrageenan. Based on broken-line analysis of percentage weight gain and feed conversion efficiency, the methionine requirements of Japanese flounder in the presence of 0.6 g kg–1 of cystine were 14.9 and 14.4 g kg–1 dry diet, respectively. After the growth study was finished, a direct estimate of methionine requirement was made by examining the influence of dietary methionine level on 14C-methionine oxidation by determining radioactive carbon dioxide, protein and nonprotein fractions of the whole body. The dose–response curve between expired radioactive CO2 and dietary methionine levels showed that the optimum methionine level for the flounder was estimated to be within the range of 14.3–17.3 g kg–1 of diet in high agreement with values obtained from the growth study.