Two healthy human females were loaded with D-phenylalanine-2′, 3′, 4′, 5′, 6′-²H (D-Phe-d₅) (8 mg/kg body weight) together with essential amino acids and a low-protein diet. Blood samples from these subjects were obtained immediately before, and at 1, 4 and 8 h after loading. D- and L-phenylalanine in the plasma were separated by high-performance liquid chromatography, and the molar ratio of Phe-d₅/Phe-d₀ in the L-isomer fraction was determined by gas chromatography-mass spectrometry with a multiple ion detector. The molar ratios of D-Phe-d₅ and L-Phe-d₅ to naturally occurring Phe-d₀ were calculated. The average minimum values of D-Phe-d₅/L-Phe-d₀ and L-Phe-d₅/L-Phe-d₀ were 0.38 (subject S: 0.16, T: 0.60) and 0.015 (S: 0.014, T: 0.015), respectively, both being obtained 1 h after loading. The average maximum values of D-Phe-d₅/L-Phe-d₀ and L-Phe-d₅/L-Phe-d₀ were 0.78 (S: 0.72, T: 0.84) and 0.043 (S: 0.045, T: 0.040), respectively, both being reached 4 h after loading. Thus, the existence of L-Phe-d₅ in plasma confirmed the conversion of D-phenylalanine to the L-form in the human body.