Despite extensive description of the damaging effects of chronic alcohol exposure on brain structure, mechanistic explanations for the observed changes are just emerging. To investigate regional brain changes in protein expression levels following chronic ethanol treatment, one rat per sibling pair of male Wistar rats was exposed to intermittent (14 h/day) vaporized ethanol, the other to air for 26 weeks. At the end of 24 weeks of vapor exposure, the ethanol group had blood ethanol levels averaging 450 mg%, had not experienced a protracted (> 16 h) withdrawal from ethanol, and revealed only mild evidence of hepatic steatosis. Extracted brains were micro-dissected to isolate the prefrontal cortex (PFC), dorsal striatum (STR), corpus callosum genu (CCg), CC body (CCb), anterior vermis (AV), and anterior dorsal lateral cerebellum (ADLC) for protein analysis with two-dimensional gel electrophoresis. Expression levels for 54 protein spots were significantly different between the ethanol- and air-treated groups. Of these 54 proteins, tandem mass spectroscopy successfully identified 39 unique proteins, the levels of which were modified by ethanol treatment: 13 in the PFC, 7 in the STR, 2 in the CCg, 7 in the CCb, 7 in the AV, and 5 in the ADLC. The functions of the proteins altered by chronic ethanol exposure were predominantly associated with neurotransmitter systems in the PFC and cell metabolism in the STR. Stress response proteins were elevated only in the PFC, AV, and ADLC perhaps supporting a role for frontocerebellar circuitry disruption in alcoholism. Of the remaining proteins, some had functions associated with cytoskeletal physiology (e.g., in the CCb) and others with transcription/translation (e.g., in the ADLC). Considered collectively, all but 4 of the 39 proteins identified in the present study have been previously identified in ethanol gene- and/or protein-expression studies lending support for their role in ethanol-related brain alterations.