Although it is generally believed that reactive oxygen species activate NF-κB, a primary oxidative stress-responsive transcription factor, it is unclear which one among these species causes NF-κB activation. Our hypothesis is that hydroxyl radical (·OH) functions as a messenger for the activation of NF-κB. Jurkat cells, macrophages and JB6 cells were used to test this hypothesis. Cr(VI), silica and ZnO were used as sources of ·OH radicals. None of these ·OH generating systems involves exogenous H2O2. Cr(VI) expressed enhanced activity in induction of NF-κB in Jurkat cells. This activation of NF-κB was decreased by a metal chelator, diethylene triaminepentaacetic acid or a H2O2 scavenger, catalase, but was increased by superoxide dismutase. Mn(II), which reacts with Cr(IV) to inhibit this metal ion-mediated ·OH generation, decreased the NF-κB activation. Sodium formate, an ·OH radical scavenger, also inhibited the NF-κB activation. Electron spin resonance measurements show that Cr(VI) was reduced by Jurket cells to Cr(IV) and Cr(V). During the reduction process, molecular oxygen was reduced to O2- and then to H2O2, which reacted with Cr(IV) and Cr(V) to generate ·OH radical. The ·OH generation correlated with the Cr(VI)-induced NF-κB activation. Similarly, silica caused NF-κB activation in macrophages via the ·OH radical-mediated reaction. This radical was generated via metal mediated reaction from H2O2, which was generated by the reduction of molecular oxygen via O2- as an intermediate during the silica-stimulated ‘respirable burst‘. Silica particles did not cause ·OH generation either in Jurket or in JB6 cells and thus did not cause any observable NF-κB activation in these cells. ZnO induced NF-κB activation in JB6 cells through the generation of ·OH resulting from light irradiation of ZnO which was measured by electron spin resonance. The results thus show that ·OH radical functions as a messenger for NF-κB activation. Antioxidants, which scavenge ·OH radical or its precursors, inhibit NF-κB activation. Metal chelators, which make metal ions incapable of generating ·OH from H2O2, inhibit activation of this transcription factor.