Although ROS can participate in modulating the activity of the transcriptional factor NF-κB and expression of NF-κB-dependent genes, the mechanisms involved and the roles of specific ROS have not been fully determined. In particular, individual ROS appear to have differing effects on NF-κB activation dependent on the cell population studied. In the present study, we examined the ability of H2O2 to affect NF-κB activation in LPS-stimulated murine neutrophils and macrophages. Exposure of bone marrow or peritoneal neutrophils to H2O2 was associated with reduced nuclear translocation of NF-κB and decreased production of the NF-κB-dependent cytokines TNF-α and macrophage inhibitory protein-2. H2O2 treatment resulted in diminished trypsin- and chymotrypsin-like proteasome activity. The degradation of IκB-α normally found in LPS-treated neutrophils was prevented when H2O2 was added to cell cultures. In contrast to the effects found in neutrophils, H2O2 did not affect chymotrypsin-like proteasomal activity or cytokine production in LPS-stimulated macrophages, even though trypsin-like proteasomal activity was reduced. These results demonstrate that the effects of H2O2 on NF-κB and proteasomal activity are cell population specific. Copyright © 2007 the American Physiological Society.