The separation and characterization of pregnenolone, its 3-sulfate and dehydroepiandrosterone in rat brains (neurosteroids) were carried out using liquid chromatography/mass spectrometry. The genins obtained from rat brains were acetylated and then derivatized withO-methylhydroxylamine to give the respective acetate-methyloximes, which were identified by comparison of their chromatographic behavior with authentic samples during liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry. Pregnenolone 3-sulfate in rat brains was also identified with an authentic sample using liquid chromatography/electrospray ionization-mass spectrometry.