BackgroundTheGABAergic neuroactive steroid (3α,5α)‐3‐hydroxy‐pregnan‐20‐one (3α,5α‐THP; allopregnanolone) has been studied during withdrawal from ethanol (EtOH) in humans, rats, and mice. Serum 3α,5α‐THPlevels decreased, and brain levels were not altered following acute EtOH administration (2 g/kg) in maleC57BL/6Jmice; however, the effects of chronic intermittent ethanol (CIE) exposure on 3α,5α‐THPlevels have not been examined. Given thatCIEexposure changes subsequent voluntary EtOH drinking in a time‐dependent fashion following repeated cycles of EtOH exposure, we conducted a time‐course analysis ofCIEeffects on 3α,5α‐THPlevels in specific brain regions known to influence drinking behavior.MethodsAdult maleC57BL/6Jmice were exposed to 4 cycles ofCIEto induce EtOH dependence. All mice were sacrificed and perfused at 1 of 2 time points, 8 or 72 hours following the final exposure cycle. Free‐floating brain sections (40 μm; 3 to 5 sections/region/animal) were immunostained and analyzed to determine relative levels of cellular 3α,5α‐THP.ResultsWithdrawal fromCIEexposure produced time‐dependent and region‐specific effects on immunohistochemical detection of 3α,5α‐THPlevels across cortical and limbic brain regions. A transient reduction in 3α,5α‐THPimmunoreactivity was observed in the central nucleus of the amygdala 8 hours after withdrawal fromCIE(−31.4 ± 9.3%). Decreases in 3α,5α‐THPimmunoreactivity were observed 72 hours following withdrawal in the medial prefrontal cortex (−25.0 ± 9.3%), nucleus accumbens core (−29.9 ± 6.6%), and dorsolateral striatum (−18.5 ± 6.0%), while an increase was observed in theCA3 pyramidal cell layer of the hippocampus (+42.8 ± 19.5%). Sustained reductions in 3α,5α‐THPimmunoreactivity were observed at both time points in the lateral amygdala (8 hours −28.3 ± 12.8%; 72 hours −27.5 ± 12.4%) and in the ventral tegmental area (8 hours −26.5 ± 9.9%; 72 hours −31.6 ± 13.8%).ConclusionsThese data suggest that specific neuroadaptations in 3α,5α‐THPlevels may be present in regions of brain that mediate anxiety, stress, and reinforcement relevant to EtOH dependence. The changes that occur at different time points likely modulate neurocircuitry involved in EtOH withdrawal as well as the elevated drinking observed afterCIEexposure.