It has been suggested that Fas-mediated apoptosis plays an important role in the pathogenesis of autoimmune thyroid diseases. Our previous studies have demonstrated that normal primary thyroid epithelial cells are resistant to Fas-mediated apoptosis, but the resistance can be overcome by pretreatment with a combination of interferon-gamma (IFN-gamma) and IL-1beta. To understand the molecular mechanism responsible for the IFN-gamma/IL-1beta effects, we profiled changes in the transcription induced by these two cytokines in normal human thyroid cells, using cDNA microarrays. We found that IFN-gamma/IL-1beta showed a significant increase in apoptosis-related genes such as inducible nitric oxide synthase (iNOS), receptor-interacting protein 2 (RIP2), and caspases 10. These increases were confirmed by other methods, including real-time PCR and Western blot. Furthermore, the sensitization of primary thyroid epithelial cells to Fas-mediated apoptosis by IFN-gamma/IL-1beta was significantly blocked by a general caspase inhibitor, z-VAD, or by the combination of two specific individual caspase inhibitors. In addition, our results showed that IFN-gamma/IL-1beta enhance p38 MAPK phosphorylation and that SB 203580, a p38 MAPK inhibitor, can inhibit IFN-gamma/IL-1beta-induced p38 MAPK phosphorylation. SB 203580 also significantly prevented cytokine-induced iNOS expression and caspase activation and thus blocked Fas-mediated apoptosis of thyroid cells sensitized by IFN-gamma/IL-1beta. In conclusion, our data suggest that both p38 MAPK and iNOS are involved in IFN-gamma/IL-1beta-induced sensitization of the thyroid cells to Fas-mediated apoptosis via the activation of caspases 3, 7, and 10 and that this pathway may be further activated by BID. This hints that inflammatory cytokines regulate death-receptor-mediated apoptosis at multiple points in the process.