To investigate dietary ascorbic acid (AA) requirement of juvenile ayu (Plecoglossus altivelis) weighing 1.27 +/- 0.02 g, eight diets were formulated with graded levels (0, 20, 40, 80, 160, 320, 640 and 1280 mg AA kg(-1)) of AA supplied as ascorbyl polyphosphate. Each experimental diet was fed to four-replicate groups to apparent satiation three times a day for 8 weeks. At the end of the feeding trial, fish fed AA-deficient diet showed visible AA deficiency signs and low survival. Based on the four-parameter saturation kinetics model, the calculated AA requirement levels for each dose-dependent response [weight gain, hepatic AA concentration, hydroxyproline (HyPro) concentration in skin and HyPro concentration in backbone] were 116, 226, 47 and 35 mg kg(-1), respectively. Based on the maximal growth performance, a level of 116 mg AA kg(-1) was recommended for commercial diet of juvenile ayu. To maintain tissue HyPro saturation and avoid AA deficiency symptoms, the minimum required dietary AA level was 47 mg kg(-1). Hepatic AA saturation was considered as the most stringent criterion for determination of AA requirement.