This study aimed to reevaluate the dietary methionine requirement by means of the plasma methionine and ammonia concentrations in surgically modified rainbow trout, Oncorhynchus mykiss. A total of 35 rainbow trout averaging 505 ± 6.5 g (initial body weight, mean ± SD) were randomly distributed into seven groups with five fish in each group. After 48 h of food deprivation, each group was fed one of seven L-amino acid-based diets containing graded levels of methionine (0.25, 0.40, 0.50, 0.60, 0.70, 0.80 or 0.95% of diet, dry matter bases) by intubation at 1% bodyweight. Blood samples were taken at 0, 5 and 24 h after intubation. Post-prandial plasma free methionine concentrations (PPmet, 5 h after intubation) and post-absorptive plasma free methionine concentrations (PAmet, 24 h after intubation) of fish fed diets containing 0.60% or more methionine were significantly (P < 0.05) higher than those of fish fed diets containing 0.50% or less methionine. PPmet and PAmet in fish fed diets containing 0.60% or higher methionine were not significantly different except the PPmet of fish fed a diet containing 0.95% methionine. Post-prandial plasma ammonia concentrations (PPA, 5 h after intubation) of fish fed diets containing 0.70% or more methionine were significantly higher than those of fish fed diets containing 0.60% or less methionine, and PPA of fish fed diets containing 0.25% and up to 0.60% methionine were not significantly different from each other. Broken-line model analyses on PPmet, PAmet, and PPA indicated that the dietary methionine requirement of rainbow trout was between 0.59% (1.69) and 0.67% (1.91) of diets (% dietary protein bases) when the diets contained 0.5% cystine.