This study was set up to investigate the mechanism of four inhibitors of interleukin-1(IL-1)-alpha and tumor necrosis factor-(TNF)alpha activated nuclear factor kappaB (NFkappaB) in whole cells. The compounds fall into two classes: the first comprised two chain-breaking antioxidants, curcumin (diferulolylmethane) and nordihydroguaiaritic acid. The second class were two thiol-modifying agents, N-ethylmaleimide (NEM) and 2-chloro-1,3dinitrobenzene (CDNB). Both sets of compounds were found to inhibit NFkappaB in tumour necrosis factor-activated Jurkat T lymphoma cells and interleukin 1-activated EL4.NOB-1 thymoma cells as determined by electrophoretic mobility shift assay using a specific NFkappaB DNA probe. In unstimulated cells the compounds were found to modify NFkappaB prior to chemical dissociation with sodium deoxycholate. They also inhibited DNA binding by NFkappaB when added to nuclear extracts from stimulated cells. Both of these effects occurred over a concentration range comparable to that which inhibited cytokine-activated NFkappaB in intact cells. All four agents were found to react directly with the p50 subunit of NFkappaB. However, only the antioxidants, curcumin and nordihydroguaiaritic acid (NDGA) were found to inhibit IkappaBalpha degradation activated by tumour necrosis factor-alpha. These results suggest that NFkappaB itself is susceptible to direct inhibition by a range of pharmacological agents. Furthermore, curcumin and nordihydroguaiaritic acid inhibit NFkappaB by interfering with IkappaBalpha degradation and reacting with p50 in the NFkappaB complex. These findings are likely to be useful in the attempt to develop agents which inhibit NFkappaB-dependent gene transcription.