In rat glial cells the lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) gene expression was enhanced by extracellular glucose concentration in a dose-dependent manner. On the other hand, 2-deoxy-d-glucose decreased the LPS-induced iNOS gene expression even in the presence of glucose (6 gm/l), suggesting that glucose metabolism is linked to the regulation of iNOS gene expression. The intracellular NADPH/NADP+ directly correlated with the extracellular glucose concentration, and the reduction of NADPH generation via a block of glucose-6-phosphate dehydrogenase (G6PD) by treatment with dehydroepiandrosterone or the antisense DNA oligomer of G6PD mRNA resulted in the inhibition of iNOS gene expression. Gel shift assays showed that CAAT/enhancing binding protein (C/EBP), rather than AP-1 or NF-kappaB, correlated better with a glucose-dependent increase in iNOS gene expression. The induction of C/EBP DNA binding activity by LPS and glucose was attributable mainly to the increase in C/EBP-delta protein. The cotransfection with wild-type C/EBP-delta increased the iNOS promoter activity to the level achieved with a higher glucose concentration in the presence of LPS. Therefore, our results suggest that C/EBP-delta may be a critical mediator in glucose-mediated regulation of iNOS gene expression.