The dietary phosphorus (P) requirement for large fish is difficult to estimate because of insensitivities of known P status indicators. We examined dietary P requirement of large rainbow trout (mean body weight 278 g) using recently identified P-responsive genes (mRNA abundances) as well as conventional serum P and bone P. Fish were fed six diets (varied P contents), and the tissues of intestine, pyloric caeca (PC), kidney, serum and bone were collected at varying time intervals. Serum P responded clearly to dietary P by day 2, but the estimated P requirement based on this variable changed as feeding duration continued. Bone P did not respond clearly until week 7. Among P-responsive genes studied, Na/Pi cotransporter in PC (PC-NaPi) was the most sensitive, and responded in 2 days. Fish-to-fish (within treatment) variance was larger in mRNA than in serum P and bone P levels. Estimated dietary P requirements (%P in dry diet) were 0.45 (based on serum P), 0.45 (based on bone P), 0.36 (based on PC-NaPi), 0.33 (based on intestinal NaPi), 0.71 (based on renal NaPi), and 0.33 (based on mitochondrial Pi carrier). This study is the first to evaluate the potential of genomic approaches in determining nutrient requirements of fish.