Abstract: The in vitro metabolism of 6-chloro -butyl phthalide(CBP)was studied bv rathepatic microsomes.The constitution of the incubation system was optimized and a RP-HPLC-DADmethod was developed for the on-line analysis of CBP and its metabelites.The difference of themetabolism profiles of CBP by rat liver microsomes induced by phenoharbital(PB)and3-methylcholanthrene(3-MC)was also compared.TLC,column chromatography and preparativeHPLC were used for the isolation and purification of the major metabotites and they were identified as(R/S) γ -hydroxy-CBP and(R/S) β-hydroxy-CBP by UV,NMR and MS.