Astroglia are important in immunocompetence and response to injury within the CNS. Activated astroglia respond, in part, by expressing inducible nitric-oxide synthase (iNOS) and subsequent catalytic production of nitric oxide. Results from a previous study in our laboratory, in the human A172 astroglial cell line, revealed that induction of iNOS activity by tumor necrosis factor-α+interferon-γ+interleukin-1β was inhibited by 24-h exposure to a high ethanol concentration (200 mM), but enhanced by 50 mM ethanol. In the work reported in this article, we tested the working hypothesis that ethanol acts transcriptionally to modulate cytokine-induced expression of the iNOS gene, NOS2A, in human astroglia. Ethanol, 50 or 200 mM, did not directly alter in vitro catalytic activity of the iNOS enzyme, indicating that ethanol does not affect the enzyme directly. Likewise, ethanol exposure after a 12-h cytokine-stimulation period had no effect on in vivo iNOS activity. However, when cells were simultaneously exposed to ethanol and cytokines for 12 h, in vivo iNOS activity was altered. That ethanol must be present during cytokine stimulation to influence iNOS activity is consistent with a transcriptional mechanism of action. In addition, steady-state expression of iNOS protein and NOS2A mRNA levels were modulated in a biphasic manner by ethanol similar to that noted previously for iNOS activity. These findings strongly support the suggestion that ethanol modulates cytokine-induced iNOS expression in A172 cells at a pretranslational site. These findings should be instrumental in the identification of the critical ethanol-sensitive elements involved in the regulation of NOS2A in human astroglia.