The role of uncoupling protein-2 (UCP-2) in β-cells is presently unclear. We have tested the notion that UCP-2 participates in β-cell defense against oxidants. Expression of the UCP-2 gene in clonal β-cells (INS-1) was decreased by 45% after 48 h of culture with vitamin E and selenite. When INS-1 cells were exposed to 200 μM H₂O₂ for 5 min, the cell viability (MTT assay) decreased to 85 ± 1, 61 ± 1, 40 ± 2, and 39 ± 2% of control when measured respectively 30 min, 2 h, 6 h, and 16 h after H₂O₂ exposure. At corresponding time points UCP-2 mRNA levels were 1.01 ± 0.09, 1.53 ± 0.15 (P < 0.05), 1.44 ± 0.18 (P = 0.06), and 1.12 ± 0.09 fold of control, i.e., transiently increased. We next tested whether overexpression of UCP-2 could enhance resistance of β-cells toward H₂O₂ toxicity. A cotransfection method using EGFP as a suitable marker and a human cDNA UCP-2 construct was used for transient overexpression of UCP-2. Transfected cells expressed the gene about 30-fold more than normal cells. After exposure to H₂O₂ (200 μm, 5 min), the survival of UCP-2 overexpressing cells was measured 30–45 min later by flow cytometry. Survival was 13 ± 0.05% higher than control (EGFP only) cells, P < 0.004 for difference. The results indicate that oxidative stress induces UCP-2 expression in β-cells, and that UCP-2 serves a role in β-cell defense against oxidative stress.