Acute ethanol administration stimulates the hypothalamic-pituitary-adrenal (HPA) axis and increases (3a,5a)-3-hydroxypregnan-20-one (3a,5a-THP) levels in rat brain and plasma. Increased 3a,5a-THP levels contribute to the anxiolytic, anticonvulsant, sedative and pro-aggressive actions of ethanol. We now explored the effects of ethanol on brain and plasma 3a,5a-THP levels in C57BL/6J (B6) and DBA/2J (D2) mice, two inbred strains with different sensitivity to behavioral effects of alcohol. Mice were injected with ethanol (2 g/kg, i.p.) or saline and were sacrificed 1 h later. 3a,5a-THP levels were measured by radioimmunoassay in cerebral cortex and by gas chromatography/mass spectrometry in plasma. Ethanol increased cerebral cortical 3a,5a-THP (+38%, P < 0.005) in D2 but not B6 mice. In contrast, plasma 3a,5a-THP levels were decreased (-27%, P < 0.01) in B6 mice, but did not change in D2 mice. These differential effects of ethanol on 3a,5a-THP levels may be due to the different genetic background of these strains. To further explore this hypothesis, we examined ethanol-induced changes in cerebral cortical 3a,5a-THP levels of some B6xD2 (BXD) recombinant inbred strains. Basal 3a,5a-THP levels across 8 BXD strains range between 1.81 and 3.72 ng/g, equivalent to a 2-fold genetic variation [F(9,79) = 6.27, P < 0.0001] and heritability of 0.40. The ethanol-induced changes in cerebral cortical 3a,5a-THP levels range between +4 and +63% and were negatively correlated (Spearman -0.82, P = 0.02) with behavioral phenotypes of ethanol consumption previously determined in the BXD strains and available in the GeneNetwork database (www.genenetwork.org). Greater ethanol intake was associated with smaller elevation of 3a,5a-THP following ethanol challenge across these BXD mice. This preliminary finding supports the hypothesis that neurosteroid responses to ethanol may be putative biomarkers of excessive alcohol consumption. Future studies will be required to further explore this hypothesis.