Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1 beta (IL-1 beta), interferon-gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1 beta and IFN gamma (IL-1 beta/IFN gamma)-induced apoptotic cell death. TNF alpha neither induced beta-cell death nor did it potentiate the effects of IL-1 beta, IFN gamma or IL-1 beta/IFN gamma . The cytotoxic effect of IL-1 beta/IFN gamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1 beta/IFN gamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1 beta/IFN gamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -3. Transduction of cells with Ad-Bcl-X-L blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X-L as a potential target to combat beta-cell apoptosis.