D-kynurenine (D-KYN), a metabolite of D-tryptophan, can serve as the bioprecursor of kynurenic acid (KYNA) and 3-hydroxykynurenine, two neuroactive compounds that are believed to play a role in the pathophysiology of several neurological and psychiatric diseases. In order to investigate the possible presence of D-KYN in biological tissues, we developed a novel assay based on the conversion of D-KYN to KYNA by purified D-amino acid oxidase (D-AAO). Samples were incubated with D-AAO under optimal conditions for measuring D-AAO activity (100 mM borate buffer, pH 9.0), and newly produced KYNA was detected by high-performance liquid chromatography (HPLC) with fluorimetric detection. The detection limit for D-KYN was 300 fmol, and linearity of the assay was ascertained up to 300 pmol. No assay interference was noted when other D-amino acids, including D-serine and D-aspartate, were present in the incubation mixture at 50-fold higher concentrations than D-KYN. Using this new method, D-KYN was readily detected in the brain, liver, and plasma of mice treated systemically with D-KYN (300 mg/kg). In these experiments, enantioselectivity was confirmed by determining total kynurenine levels in the same samples using a conventional HPLC assay. Availability of a sensitive, specific, and simple method for D-KYN measurement will be instrumental for evaluating whether D-KYN should be considered for a role in physiology and pathology.