In previous studies, changes in adult ethanol intake after early environmental experiences, such as short and prolonged maternal separation, have been described in male rats. The aim of this study was to further investigate long-term effects of maternal separation on voluntary ethanol intake as well as brain opioid and nociceptin/orphanin FQ (N/OFQ) peptides in female Wistar rats. During postnatal days (PNDs) 1-21, rat pups were subjected to 15 min (MS15) or 360 min (MS360) of daily maternal separation, or were kept under normal animal facility rearing (AFR) conditions. At 10 weeks of age, ethanol intake was measured using a two-bottle free choice paradigm and dynorphin B (DYNB), Met-enkephalin-Arg6-Phe7 (MEAP) and N/OFQ levels were analyzed. MS15 resulted in changes in hypothalamus (DYNB), medial prefrontal cortex and nucleus accumbens (MEAP), and amygdala (N/OFQ). MS360 induced alterations in medial prefrontal cortex (MEAP) and amygdala (N/OFQ). MS15 and MS360 had no effect on ethanol intake. However, 4 weeks of ethanol intake affected peptide levels differently in MS15, MS360 and AFR rats and resulted in attenuation of the separation-induced differences. These results show that even though maternal separation has no effect on voluntary ethanol intake in female rats, the ethanol-induced effects on peptide levels depend on the early environmental setting.