This study was undertaken to investigate: (1) the effects of both deficiencies and excesses in essential amino acids (EAAs) from an estimated optimum dietary EAA pattern on nitrogen (N) utilization and excretion of rainbow trout Oncorhynchus mykiss, (2) the effects of dietary digestible protein (PD): digestible energy (ED) ratio (PD:ED) on N utilization and excretion of O. mykiss and (3) the potential interaction of these two factors. A 3 × 3 factorial experiment was conducted, with the two factors EAA pattern and PD:ED ratio. The three levels of EAA pattern were: (1) optimum EAA pattern, (2) 60% deficiencies in the three amino acids arginine, histidine and lysine, and (3) 60% excesses in the three amino acids arginine, histidine and leucine. The three levels of PD:ED ratio were 18, 21 and 24 g MJ−1. Amino acid deficiencies from an optimum amino acid pattern caused reductions in mean N retention of 29 to 37%, with the greatest reduction associated with the lowest PD:ED ratio, and similar substantial increases in total N and ammonia-N excretion at all of the dietary PD:ED ratios investigated. Amino acid excesses, however, did not negatively affect N retention or excretion. Increasing PD:ED ratio was associated with decreasing N retention and increasing N excretion over the range of dietary protein and lipid levels tested. Results of this study showed that a diet with optimum dietary amino acid pattern and lowest PD:ED ratio produced the highest N retention (47% of ingested N) and the lowest total N and ammonia-N excretion of O. mykiss.