The pharmacological profile of allopregnanolone, a neuroactive steroid that is a potent positive modulator of gamma-aminobutyric acidA (GABAA) receptors, is similar to that of ethanol. Recent findings indicate that acute injection of ethanol increased endogenous allopregnanolone to pharmacologically relevant concentrations in male rats. However, there are no comparable data in mice, nor has the effect of ethanol drinking on endogenous allopregnanolone levels been investigated. Therefore, the present studies measured the effect of ethanol drinking and injection on allopregnanolone levels in male and female C57BL/6 mice. One group was given 17 days of 2-h limited access to a 10% v/v ethanol solution in a preference-drinking paradigm, while another group had access to water only. The ethanol dose consumed in 2 h exceeded 2 g/kg. Then, separate groups of mice were injected with either 2 g/kg ethanol or saline. Mice were killed 30 min after the 2-h drinking session or injection. Blood ethanol concentration was significantly higher in the ethanol-injected versus ethanol-drinking groups, even though the dose was similar. Consumption of ethanol significantly increased brain allopregnanolone levels in male but not female mice, compared with animals drinking water, but did not alter plasma corticosterone levels. In contrast, injection of ethanol did not significantly alter brain allopregnanolone levels in male or female mice and only significantly increased plasma corticosterone levels in the male mice, when compared with saline-injected animals. The sex differences in the effect of ethanol administration on endogenous allopregnanolone levels suggest that the hormonal milieu may impact ethanol's effect on GABAergic neurosteroids. Importantly, these data are the first to report the effect of ethanol drinking on allopregnanolone levels and indicate that ethanol consumption and ethanol injection can produce physiologically relevant allopregnanolone levels in male mice. These results have important implications for studies investigating the potential role of endogenous allopregnanolone levels in modulating susceptibility to ethanol abuse.