Purpose: 99mTc-HMPAO is a radiopharmaceutical for imaging cerebral blood flow. HMPAO (RR, SS)-4,8- diaza-3,6,6,9-tetramethylundecan-2,10- dione bisoxime) has three stereoismers such as, meso-, d-, and l-HMPAO. Techentium complexes of meso-HMPAO and d,l-HMPAO are known to have different in vivo brain uptakes. In this study, enantiomers of HMPAO (d-HMPAO and l-HMPAO) were separated from d,l-HMPAO. These enantiomers were labeled with 99mTc and the biodistribution studies were performed in mice. Materials and Methods: An intermediate imine product was produced from 2,3-butanedione monooxime and 2,2-dimethyl- 1,3-propanediamine (54% yield) and was reduced into a mixture of three isomers (35% yield). The meso-isomer was separated from d,l-mixture by repeated fractional crystallization (11% yield). The d- and l-enantiomers were subsequently separated by co-crystallization with optical isomers of tartaric acid (25% and 5% yield, respectively). Each enantiomeric HMPAO was labeled with 99mTc by reacting with SnCl2¡¤2H2O and 99mTc-pertechnetate. Biodistribution study was performed 1 hr after tail vein injection to ICR mice. Results: Radiochemical purities of each compound were over 80%. In biodistribution study, the brain uptakes of d,l- d- and l-form were 1.34, 1.12 and 1.67% ID/g, respectively. In case of l-Isomer the brain uptake was higher (1.5 fold) than d-isomer. Conclusion: We successfully purified each enantiomeric HMPAO. In biodistribution study of stereoismers of 99mTc-HMPAO in mice, l-HMPAO may show better brain image than d,l-HMPAO which was supplied in a commercial kit. (Korean J Nucl Med 2001;35:69-74)