An 8-week feeding trial was conducted to determine dietary isoleucine (Ile) requirement of rainbow trout, Oncorhynchus mykiss fingerlings. Six isonitrogenous (450 g kg(-1)) and isocaloric (20.90 kJ g(,)(-1) gross energy) diets were designed with varied Ile levels (6.0-18.5 g kg(-1), dry diet). Twenty fish (1.66 +/- 0.02 g/fish; 5.25 +/- 0.34 cm/fish) were distributed in triplicate groups in 75-L troughs attached to flow-through system (2-2.5 L minute(-1)) and were fed twice daily (09:00 and 17:00 hours) at 5% BW day(-1). Maximum live weight gain (LWG %), specific growth rate (SGR), protein efficiency ratio (PER) and the best-feed conversion ratio (FCR) were observed at 13.5 g kg(-1) Ile fed diet. Quadratic regression analysis of growth data predicts optimum Ile requirement to be at 13.73 (LWG %), 13.41 (FCR), 13.44 (PER) and 13.35 (BPD) g kg(-1) respectively. Highest (p < 0.05) protein, lowest moisture and intermediate fat contents were noted at 13.5 g kg(-1) Ile fed diet. Haematological variables such as haemoglobin (Hb), haematocrit (Hct) and red blood cell (RBC) count also showed highest values at 13.5 g kg(-1) Ile diet. Plasma cholesterol, triglycerides, glucose and urea did not produce any significant (p > 0.05) changes among the groups, while as alanine transaminase (ALT), aspartate transaminase (AST) and uric acid levels showed significant (p < 0.05) variations. Highest total protein content and lowest alkaline phosphatase (ALP) were observed at 13.5 g kg(-1) Ile fed diet, while albumin and globulin content did not produce any significant (p > 0.05) difference among the groups. Significant (p < 0.05) differences were also observed in intestinal enzymatic activities and antioxidant status. Highest (p < 0.05) TOR and 4E-BP mRNA gene expression level were also found at 13.5 g kg(-1) Ile fed diet. On the basis of above data, it is recommended that 13.48 g kg(-1) Ile, corresponding to 29.95 g kg(-1) of dietary protein, would be useful for optimum growth and best nutrient conversion of this species.