Reactive oxygen species (ROS) and nitric oxide (NO) are proposed mediators of cytokine-induced β-cell destruction in type 1 diabetes. We produced transgenic mice with increased β-cell expression of manganese superoxide dismutase (MnSOD) and catalase. Expression of these antioxidants increased β-cell ROS scavenging and improved β-cell survival after treatment with different sources of ROS. MnSOD or catalase conferred protection against streptozotocin (STZ)-induced β-cell injury. Coexpression of MnSOD and catalase provided synergistic protection against peroxynitrite and STZ. To determine the potential effect of these antioxidants on cytokine-induced toxicity, we exposed isolated islets to a cytokine mixture, including interleukin-1β and interferon-γ. Cytokine toxicity was measured as reduced metabolic activity after 6 days and reduced insulin secretion after 1 day. Cytokines increased ROS production, and both antioxidants were effective in reducing cytokine-induced ROS. However, MnSOD and/or catalase provided no protection against cytokine-induced injury. To understand this, the nuclear factor-κB (NF-κB) signaling cascade was investigated. Antioxidants reduced NF-κB activation by ROS, but none of the antioxidants altered activation by cytokines, as measured by inhibitor of κB phosphorylation, NF-κB translocation, inducible NO synthase activation, and NO production. Our data agree with previous reports that antioxidants benefit β-cell survival against ROS damage, but they are not consistent with reports that antioxidants reduce cytokine toxicity. ROS appear to have no role in cytokine toxicity in primary β-cells.