This study examined influences of ethanol exposure on phosphorylation of cAMP response element-binding protein (CREB) and CRE-binding activity in the striatum of rats. The phosphorylated form of CREB increased 180% during acute intoxication, compared to sham conditions. After chronic ethanol exposure, induction of CREB phosphorylation by an acute ethanol challenge was markedly attenuated (50%) compared with acute ethanol exposure in the pair-fed condition. Gel retardation assays with oligomers encoding the rat proenkephalin CRE-1 and CRE-2 were performed to determine the effects of ethanol on CRE-binding activity. Supershift experiments demonstrated that striatal nuclear protein contains CREB and CEBPbeta (CCAAT/enhancer-binding protein) and that both transcription factors are involved in specifically binding to the DNA sequence. Rechallenging rats that had been chronically exposed to ethanol with an acute ethanol challenge reduced the CRE/nuclear protein complexes. However, supershift analyses did not show that chronic ethanol exposure altered the dimerization patterns of CREB and CEBPbeta within the complexes. Associated with these impairments in CREB binding and CREB phosphorylation was a significant reduction in two CREB-modulated factors, proenkephalin and c-fos expression. In summary, acute ethanol exposure activates the phosphorylation of CREB. Neuroadaptation to chronic ethanol exposure includes alterations in CREB physiology that may impair genes that are dependent upon CREB for transcriptional activation.