Alcoholism is characterized by tolerance, dependence, and unrestrained craving for alcohol. Adaptive responses, including changes in gene expression in neurons, are thought to account for some of these complex behavioral abnormalities. We have shown in the NG108-15 neuroblastoma x glioma hybrid cell line that ethanol increases cellular cAMP levels via activation of adenosine A(2) receptors, leading to phosphorylation of the cAMP response element-binding protein (CREB). However, phosphorylation of CREB is not sufficient to activate cAMP response element (CRE)-mediated gene expression. Here we investigate whether ethanol increases CRE-mediated gene expression via endogenous CREB using a CRE-regulated luciferase reporter construct, transfected into NG108-15 cells. We find increased luciferase activity as a function of time of exposure to ethanol. Coexpression of a dominant-negative CREB construct blocked ethanol-stimulated CRE-luciferase expression, further suggesting that CREB is required for this response. We also determined whether ethanol-induced increases in gene expression are mediated by ethanol-induced increases in extracellular adenosine. We found that CRE-mediated gene expression induced by ethanol occurs in two phases: an early phase (4 h), in which adenosine receptor blockade prevents ethanol-induced gene expression, and a later phase (14 h), which is not blocked by an adenosine receptor antagonist. In both phases, inhibition of cAMP-dependent protein kinase A (PKA) activity prevented ethanol-induced CRE-mediated luciferase expression. Our data suggest that ethanol induces cAMP-dependent gene expression regulated by CREB and PKA and that this signaling pathway may mediate some of the addictive behaviors underlying alcoholism.