A new analytical method has been developed for simultaneous quantitation of levomepromazine and its five main metabolites in serum and urine. The method uses C-2 bonded phase extraction and reversed-phase high-performance liquid chromatography, based on ion-pair formation with dodecyl sulfate. The detection limits were 15 nM for levomepromazine and N-desmethyl levomepromazine, 28 nM for levomepromazine sulfoxide, and 56 nM for 3-hydroxylevomepromazine. 7-hydroxylevomepromazine, and O-desmethyllevomepromazine in serum, and lower in urine. The method was applied to measure steady-state serum and urine concentrations of levomepromazine and metabolites in five psychiatric patients. The concentrations of levomepromazine sulfoxide and N-desmethyllevomepromazine were generally higher than the concentrations of levomepromazine. The hydroxylated and O-demethylated metabolites were also found in higher concentrations than levomepromazine, but mainly as conjugates.