The in vitro biotransformation of 6-methoxy butyl phthalide (MBP) by phenobarbital induced rat liver microsomes was investigated by GC/MS and GC/MS with TMS derivatization. Three major metabolites of MBP were identified as 3-OH-MBP, 6-OH-butyl phthalide and gamma-OH-MBP. A less polar metabolite was tentatively identified as an epoxidation product.