Cytokines have been implicated in pancreatic beta-cell destruction leading to type 1 diabetes. Exposure to interleukin-1beta (IL-1beta) of pancreatic beta-cells induces expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent formation of nitric oxide (NO) and prostaglandin E2 (PGE2) may impair beta-cell function. Using NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA), we have further investigated the relation between NO formation and COX-2 expression. IL-1beta stimulated the formation of NO and PGE2 by pancreatic beta-cells. L-NMMA completely inhibited IL-1beta-induced NO formation and attenuated PGE2 production. COX-2 gene transcription level and protein expression were determined by real-time PCR, Western blot and luciferase analysis. L-NMMA inhibited IL-1beta-induced promoter activity, gene transcription and protein expression of COX-2 in pancreatic beta-cells. Therefore, we concluded that NO-affected COX-2 activity is directly linked to COX-2 gene transcription and protein expression in pancreatic beta-cells. The identification of a novel interaction of NO on the COX signaling pathway in beta-cells provides a strategy of intervention for further evaluating the role of NO and PGE2 in autoimmune diabetes.