Neuroactive steroids are endogenous modulators of neuronal activity that modulate central nervous system inhibitory tone and influence motivation and emotional behaviors. The inhibitory neuroactive steroid (3a,5a)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) is a potent positive modulator of γ-aminobutyric acid type A (GABAA) receptors. In rats, systemic ethanol administration increases 3α,5α-THP in the blood plasma, cerebral cortex, and hippocampus. Ethanol-induced increases of 3α,5α-THP contribute to the behavioral and neurophysiological effects of ethanol in rats and the subjective effects of alcohol in humans. Previous in vivo studies suggest that ethanol-induced increases of 3α,5α-THP are dependent on the adrenal glands, but in vitro studies suggest ethanol produces local synthesis of 3α,5α-THP. Furthermore, limitations in steroid assays have prevented the study of ethanol-induced changes of 3α,5α-THP in many of the brain regions implicated in alcoholism. The first aim of this project used 3α,5α-THP immunohistochemistry (IHC) to determine ethanol effects on cellular levels of 3α,5α-THP across the rat brain, and determine the role of the adrenal glands in these effects. We showed that ethanol produces divergent, brain region specific, effects on cellular 3α,5α-THP levels. We also showed that ethanol can increase or decrease local iv brain levels of 3α,5α-THP independent of adrenal activation in subcortical brain regions, but increases of 3α,5α-THP in the medial prefrontal cortex (mPFC) are dependent on the adrenal glands. Previous studies have shown that systemic 3α,5α-THP can reduce ethanol reinforcement and consumption. However, systemic 3α,5α-THP is metabolized rapidly and can produce sedation, limiting therapeutic value. Therefore, in aim 2 we used gene delivery to drive local neuroactive steroid synthesis in the nucleus accumbens (NAc) or ventral tegmental area (VTA), which are both implicated in ethanol reinforcement and consumption. We showed that adeno-associated viral vector-mediated local increases of 3α,5α-THP in the VTA were associated with a long-term reduction in ethanol reinforcement and consumption. Further investigation showed that 3α,5α-THP is located in tyrosine hydroxylase (TH) positive and negative neurons, but not astrocytes in the VTA. These studies showed that ethanol can produce divergent effects on local brain levels of 3α,5α-THP and increasing local levels of 3α,5α-THP in the VTA is associated with reduced ethanol reinforcement and consumption.