The purpose of this study was to evaluate the toxicity of L-phenylalanine when administered daily in the diet to rats for at least 28 days. Male and female Crl:CD®(SD)IGS BR rats were assigned to four groups. Each group received diets containing basal diet or 0.5, 1.5, or 5.0% (w/w) L-phenylalanine. There were no clinical or ophthalmic observations that were considered to be related to L-phenylalanine. Effects of L-phenylalanine administration were noted in mean body weights and mean body weight gains in females fed 0.5% and in males and females fed 5.0% (w/w) L-phenylalanine diets. Effects were also noted in mean food consumption in males and females fed the 5.0% (w/w) L-phenylalanine diet. The lower food consumption and body weights of the males and females fed the 5.0% L-phenylalanine diet were considered to be signs of mild toxicity. Administration of L-phenylalanine at a dose of 5.0% of the diet was associated with mildly increased red blood cell count and mildly decreased mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and glucose in females. There were no L-phenylalanine-related toxic changes for organ weight, or macroscopic or microscopic findings. In conclusion, the no-observed-effect level (NOEL) of dietary exposure of male rats to L-phenylalanine is 1.5% (w/w) L-phenylalanine. The NOEL of dietary exposure of female rats to L-phenylalanine is less than 0.5% (w/w) L-phenylalanine. However, the no-observed-adverse-effect level (NOAEL) for males and females is 1.5% (w/w) L-phenylalanine.