The objective of the present study was to evaluate the validity of a FFQ in the estimation of dietary amino acid intake. Amino acid intake was calculated using a composition database developed based on the Standard Tables of Food Composition for amino acids in Japan. Subjects were subsampled from two populations of the Japan Public Health Center-based Prospective Study who volunteered to participate in the validation study of the FFQ. The first group was from the population the FFQ was developed for (internal population; n 215) and the second was a separate population to confirm external validity (external population; n 350). The validity of the FFQ was evaluated using 28 d weighed dietary records (DR) as a reference method. Spearman's rank correlation coefficients (CC) between amino acid intake from the FFQ and DR were calculated. The dietary intake of amino acids was slightly underestimated by the FFQ. Deattenuated CC of energy-adjusted amino acid intake according to the FFQ and corresponding amino acid intake according to the DR ranged from 0.15 to 0.52. The median CC for twenty amino acids were 0.33 for men and 0.25 for women in the internal population, and 0.40 for men and 0.30 for women in the external population. In conclusion, the validity of the FFQ in estimating amino acid intake was low to moderate.