The analysis of stress-induced changes in the brain neurosteroid levels by liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization-mass spectrometry (ECAPCI-MS) is described. In the present method, neurosteroids were derivatized with a highly electron-affinitive reagent, 2-nitro-4-trifluoromethylphenylhydrazine (NFPH), to convert them to the corresponding hydrazones. The derivatized steroids showed over a 20-fold higher sensitivity in ECAPCI-MS than intact steroids measured by positive atmospheric pressure chemical ionization (APCI)-MS. Application of this method to the analysis of rat brain samples confirmed the significant increase in the levels of pregnenolone (PREG), progesterone (PROG), 5alpha-dihydroprogesterone (DHPROG), allopregnanolone (3alpha-hydroxy-5alpha-pregn-20-one; AP), and epiallopregnanolone (3beta-hydroxy-5alpha-pregn-20-one; EpiAP) in the fixated rats. The din stress, which we examined as a new short-term mental stress model, also elevated the brain neurosteroid levels. It is known that various types of stress lower the gamma-aminobutyric acid type A (GABA(A)) receptor function and induce the neuronal overexcitation. The increase in the brain level of AP, a potent positive modulator of GABA(A) receptors, may be the defensive response against acute stress. The increase in the brain concentration of its precursors, PREG, PROG, and DHPROG, may be associated with the acceleration of the AP synthesis. Thus, the present studies suggest that changes in the brain levels of neurosteroids may play an important role in the homeostatic mechanisms that counteract the inhibitory effect of stress on the GABA(A) receptor function.