OBJECTIVE - The unfolded protein response (UPR) is a conserved cellular response designed to alleviate damage and promote survival of cells experiencing stress; however, prolonged UPR activation can result in apoptotic cell death. The UPR, activated by cytokine-induced nitric oxide (NO) production, has been proposed to mediate β-cell death in response to cytokines. In this study, the role of UPR activation in cytokine-induced β-cell death was examined. RESEARCH DESIGN AND METHODS - The effects of cytokine treatment of rat and human islets and RINm5F cells on UPR activation, NO production, and cell viability were examined using molecular and biochemical methodologies. RESULTS-UPR activation correlates with β-cell death in interleukin (IL)-1-treated rat islets. NO mediates both cytokine-induced UPR activation and β-cell death as NO synthase inhibitors attenuate each of these IL-1-stimulated events. Importantly, cytokines and tunicamycin, a classical UPR activator, induce β-cell death by different mechanisms. Cell death in response to the classical UPR activator is associated with a 2.5-fold increase in caspase-3 activity, while IL-1 fails to stimulate caspase-3 activity. In addition, cell death is enhanced by ∼35% in tunicamycin-treated cells expressing an S51A eIF2α mutant that cannot be phosphorylated or in cells lacking PERK (protein kinase regulated by RNA/endoplasmic reticulum-like kinase). In contrast, neither the absence of PERK nor the expression of the S51A eIF2α mutant affects the levels of cytokine-induced death. CONCLUSIONS - While cytokine-induced β-cell death temporally correlates with UPR activation, the lack of caspase activity and the ability of NO to attenuate caspase activity suggest that prolonged UPR activation does not mediate cytokine-induced β-cell death.