Interleukin (IL)-1 beta-induced inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans is mediated in part by nitric oxide (NO). The NO synthase cofactor 5,6,7,8-tetrahydrobiopterin (BH(4)) supports NO synthesis in many cell types and IL-1 beta-induced NO generation and inhibition of insulin secretion have been previously correlated with intracellular BH(4 )levels in rat insulinoma cells. Using rat islets and the beta cell line BRIN-BD11, we have investigated whether synthesis of BH(4) limits IL-1beta-induced NO generation and inhibition of glucose-induced insulin secretion. IL-1 beta-induced NO generation by BRIN cells and islets was reduced by 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of de novo BH(4) synthesis. Sepiapterin, the substrate for salvage pathway BH(4) synthesis, reversed this inhibitory effect of DAHP in islets but not BRIN cells. DAHP reversed IL-1 beta-induced inhibition of islet insulin secretion, an effect prevented by sepiapterin. We conclude that BH(4) generation is necessary for IL-1 beta-induced NO generation in rat islets and BRIN cells. While a contribution of non-NO mediators cannot be excluded, our results support the proposal that IL-1 beta-induced, NO-mediated inhibition of insulin secretion in rat islets is dependent on the NOS cofactor BH(4).