A sensitive and selective method for the determination of four threonine (Thr) isomers (L-Thr, D-Thr, L-allo-Thr and D-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of D-Thr, D-allo-Thr and L-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol-5 pmol per injection, and that of L-Thr is 50 fmol-50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of D-Thr, D-allo-Thr and L-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of D-Thr and D-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of D-Thr (0.85 +/- 0.05 nmol/g wet tissue) and D-allo-Thr (5.01 +/- 0.32 nmol/g wet tissue) were found in the corpus striatum. L-allo-Thr was not present in any of the tested tissues and physiological fluids.