Nicotinamide and serotonin are synthesized from L-tryptophan in mammals. It is important to know the nutritional factors affecting the synthesis of nicotinamide and serotonin. We investigated the effects of amino acid composition. Young adult rats were fed ad libitum for 21 d a low-protein (9% casein) diet([1] control), or one of the low protein diets supplemented with following amino acids: [2] glycine, L-threonine, and L-cystine, [3] L-threonine and L-cystine, [4] glycine and L-cystine, and [5] glycine and L-threonine. The amounts of glycine, L-threonine and L-cystine supplementations were 2%, 0.078%, and 0.2%, respectively, and the amino acid contents of all diet were adjusted with supplementation of L-glutamic acid. The body weight gain, food efficiency ratio, and the amino acid nutrition biomarker, which is the urinary excretion ratio of (N¹-methyl-2-pyridone-5-carboxamide+N¹-methyl-4-pyridone-3-carboxamide)/N¹-methylnicotinamide, improved by adding the amino acids glycine, L-threonine and L-cystine to a 9% casein diet. The conversion percentage of L-tryptophan to nicotinamide decreased with the addition of the amino acids glycine, L-threonine and L-cystine to a 9% casein diet, while the concentrations of serotonin in the brain, stomach and small intestine were not affected at all. The effects of each amino acid on body weight gain and the conversion ratios were also investigated. Glycine did not affect these variables. L-Cystine improved the body weight gain, the food efficiency ratio and the urine ratio, and decreased the conversion percentage. L-Threonine did not affect body weight gain or food efficiency ratio; however, it improved the urine ratio and decreased the conversion percentage.