Neurosteroids are modulators of neuronal function that may play important roles in brain maturation. We determined whether chronic prenatal ethanol exposure altered neurosteroid levels in the developing brain. Rat dams were exposed to: (i) a 5% ethanol-containing liquid diet that produces peak maternal blood alcohol levels near the legal intoxication limit (approximately 0.08 g/dL); (ii) an isocaloric liquid diet containing maltose-dextrin instead of ethanol with pair-feeding; (iii) rat chow ad libitum. Neurosteroid levels were assessed in offspring brains using radioimmunoassay or gas chromatography-mass spectrometry techniques. A prenatal ethanol exposure-induced increase in pregnenolone sulfate levels, but not dehydroepiandrosterone sulfate levels, was evident at the earliest time point studied (embryonic day 14). This effect lasted until post-natal day 5. Levels of other neurosteroids were assessed at embryonic day 20; pregnenolone levels, but not allopregnanolone levels, were elevated. Pregnenolone sulfate levels were not altered in the maternal brain. Neither pregnenolone nor pregnenolone sulfate levels were significantly altered in the fetal liver, placenta and maternal blood, indicating that the effect of ethanol is not secondary to accumulation of peripherally-produced steroids. Fetal ethanol exposure has been shown to decrease both cellular and behavioral responsiveness to neurosteroids, and our findings provide a plausible explanation for this effect.