Alcohol consumption has multiple effects in the central nervous system (CNS). Whereas, alcohol is an immunosuppressive drug the effect of alcohol on the neuroimmune system, remains unclear. In cultured astrocytes, prolactin (PRL) induces mitogenesis and the expression of inflammatory cytokines, including tumor necrosis factor-โฃ (TNFโฃ). We have recently shown that whereas ethanol does not inhibit PRL receptor binding, it markedly inhibits PRL-induced mitogenesis and TNFโฃ secretion in cultured astrocytes. It is clear that PRL activates the tyrosine phosphorylation of several proteins, including members of a novel family of protein tyrosine kinases, the Janus Kinases (JAKs). The aims of this study were to characterize PRL-induced activation of the JAK/STAT (signal transducers and activators of transcription) pathway, and to determine if ethanol affects JAK/STAT activation in cultured astrocytes. We found that PRL specifically increases the tyrosine phosphorylation of JAK2, but not JAK1, JAK3, or Tyk2, and the subsequent phosphorylation of STAT1โฃ, STAT5a, and STAT5b. Preincubation of astrocytes with ethanol markedly inhibited phosphorylation of JAK2, STAT1โฃ, STAT5a, and STAT5b. In PRL-stimulated astrocytes, ethanol inhibited binding of nuclear proteins to oligonucleotides corresponding to the gamma-interferon activated sequence (GAS). Further, ethanol blocked PRL-induced increases in interferon regulatory factor-1 (IRF-1) mRNA, a PRL/cytokine inducible transcription factor involved in the regulation of a number of cytokine inducible genes. The inhibition of tyrosine phosphorylation by ethanol was not a general effect, however, as we found that ethanol increased basal and NGF-induced tyrosine phosphorylation of extracellular signal-activated protein kinase-1 (ERK-1). These data indicate that ethanol inhibits PRL-induced tyrosine phosphorylation of the JAK/STAT pathway resulting in decreased nuclear GAS DNA binding and inhibition of the PRL inducible gene, IRF-1. Thus, suggesting that ethanol-induced inhibition of JAK2 phosphorylation may be one mechanism though which ethanol could alter the brain's response to injury or infection.