We have examined the regulation of inducible nitric oxide synthase (iNOS) gene expression by ethanol in monolayers of DLD-1 cells, an epithelial cell line derived from human intestinal adenocarcinoma. Optimum induction of iNOS mRNA in these cells was obtained with IFN-gamma and IL-1beta treatment, while further addition of TNF-alpha did not have significant effect. In a set of experiments to study ethanol effects, DLD-1 monolayers were pretreated with ethanol for 24 h and were then treated with IFN-gamma + IL-1beta for an additional 24 h. Cells pretreated with ethanol showed decreased iNOS mRNA levels, indicating that ethanol may inhibit cytokine-induced iNOS transcription or affect mRNA destabilization. The suppression was ethanol-dose dependent with an IC50 of 50 mM. In another set of experiments to study ethanol effects, DLD-1 monolayers were pretreated with 66 mM ethanol for 24 h. These cells showed significant upregulation of IL-1beta mRNA and protein as detected in the supernatants. Aliquoted supernatants from these cells (i.e., conditioned media) were added to naive DLD-1 monolayers together with IFN-gamma. Conditioned medium from ethanol-treated cells increased the IFN-gamma-induced iNOS mRNA of naive cells by threefold. Two different effects of ethanol are now reported: (a) ethanol inhibits IFN-gamma + IL-1beta-induced iNOS mRNA of the same DLD-1 cells and (b) ethanol induces cellular paracrine signals by releasing IL-1beta into the medium, which in combination with IFN-gamma increases iNOS mRNA levels of the recipient naive DLD-1 cells. Because IFN-gamma and IL-1beta are produced by intestinal immune cells, these findings may have implications for differential in vivo regulation of epithelial iNOS genes by ethanol, depending on the inflammatory and immune status of the host.