Acute treatment with extracts of Hypericum perforatum, the common plant usually called St. John's Wort, reduces voluntary ethanol intake in Marchigian Sardinian alcohol-preferring (msP) rats and acts synergistically with opioid receptor antagonists to further attenuate ethanol consumption. The present study evaluated the effect of chronic (once a day for 12 days) intragastric administration of a CO2 Hypericum perforatum extract (HPCO2), given alone or combined with naltrexone (NTX), on ethanol intake offered 2h/day in msP rats. Chronic treatment with HPCO2 markedly reduced ethanol intake at the dose of 125, but not at 7 mg/kg; the effect of 125 mg/kg was observed since the first day of treatment and remained constant across the 12 days. The same dose of HPCO2 slightly reduced the simultaneous intake of food only on day 3 and day 11 of treatment. Treated rats promptly recovered baseline ethanol intake when treatment did not precede access to ethanol (on day 8) or after the end of treatment (day 13 and day 14), suggesting that HPCO2 administrations did not induce conditioned aversion to alcohol. Chronic intraperitoneal treatment with NTX reduced ethanol intake at 3, but not at 0.5mg/kg. The synergistic effect on ethanol intake of HPCO2 and NTX was evident also in conditions of chronic treatment. HPCO2, 7 mg/kg, and NTX, 0.5mg/kg, evoked a pronounced and statistically significant reduction of ethanol intake, while being inactive. The effect on ethanol intake of the combined treatment remained stable over the 12 days of treatment; food intake was slightly reduced only on day 3 and on day 7 in response to 125 mg/kg of HPCO2 combined with NTX 0.5mg/kg, but no difference in body weight between controls and treated rats was observed at the end of treatment. Following 12-day treatment with 125 mg/kg of HPCO2, no difference was observed in the responsivity of msP rats to the effect on ethanol intake of several doses of the extract. In conclusion, the present results provide evidence for a selective and pronounced effect of HPCO2, alone or combined with naltrexone, on ethanol intake in conditions of chronic treatment, without development of tolerance. These findings further support the view that clinical trials for extracts of Hypericum perforatum in the treatment of alcoholism should be considered.