Abstract: AIM　To separate propafenone enantiomers in rat liver microsomal incubates and investigate probable stereoselectivity in their N-dealkylation. METHODS　The concentration of each enantiomer in rat liver microsomal incubates was determined through precolumn derivatization with 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate (GITC), followed by RP-HPLC assay. RESULTS　A baseline separation of propafenone enantiomers was achieved on C₁₈-ODS column, with methanol — water — glacial acetic acid (67∶33∶0.05) as mobile phase. The assay was linear from 0.5 to 320 μg*mL^-1 for each enantiomer, and the limit of detection was 100 ng*mL^-1. The method affords the average recoveries of 77.1% for R-propafenone and 76.0% for S-propafenone. Stereoselectivity was observed for the phase I metabolism of racemic propafenone in dexamethasone, β-naphthoflavone induced rat liver microsomal incubates,but not in controls. CONCLUSION　The method is simple, cheap and can be applied to study the metabolism of R- and S-propafenone in vitro. Stereoselectivity exists in their N-dealkylation.