Abstract: In the present study,a specific and reliable method was developed for thedetermination of omeprazole and its two major metabolites:hydroxyomeprazole(OH -OPZ)andomeprazole sulfone(OPZ-SFN)in Chinese adult human liver microsome by reversed phase HPLCassay. Formation of these metabolites is linear for at least 60 min and between 0.25 and 1mg·ml^-1of microsomal protein. The enzyme kinetic analysis of the reaction revealed that the hydroxylationVmax and Km obtained with the human liver microsomal preparation were 42.9 nmol·min^-1·(mgprotein)-1 and 6.49μmol·L^-1,respectively.The maximum formation rates of OPZ-SFN(Vmax)was 6.63 nmol· min^-1·(mg protein)-1, with a Km value of 11.8 μmol·L^-1. A number ofcompounds were tested for their ability to inhibit OPZ metabolism, Our results showed thatmephenytoin,diazepam and nordiazepam are competitive inhibitors and papaverine is an uncompetitiveinhibitor of OPZ hydroxylation, These studies suggest that the same isozyme metabolising MP,DZ andNDZ(may be P450 2C or P450 3A) may be involved in the hydroxylation of OPZ,Moreover,thecompounds tested also have some effects on the for mation of OPZ-SFN in vitro with Chinese humanliver microsomes.