Chiral column liquid chromatography and enantiospecific enzymatic hydrolysis were utilized to separate the enantiomers of α‐ and β‐dihydrotetrabenazine and α‐9‐O‐desmethyldihydrotetrabenazine, three benzo[a]quinolizines derived from the amine‐depleting drug tetrabenazine. An X‐ray crystal structure analysis of (−)‐α‐9‐O‐desmethyldihydrotetrabenazine gave an absolute structure of that compound as the 2S, 3S, 11bS isomer. Therefore, (−)‐α‐dihydrotetrabenazine also has the 2S, 3S, 11bS absolute configuration. (+)‐α‐Dihydrotetrabenazine, the single biologically active isomer from the metabolic reduction of tetrabenazine, thus has the absolute configuration of 2R, 3R, 11bR. For further in vitro and in vivo studies of the vesicular monoamine transporter, it is now possible to use the single enantiomer of radiolabeled α‐dihydrotetrabenazine. Chirality 9:59–62, 1997. © 1997 Wiley‐Liss, Inc.