Nitric oxide (NO) regulates cellular function, in part, by S-nitrosylating active site thiol groups of proteins. Ex vivo S-nitrosylation of NF-κB p50 significantly decreases its capacity for DNA binding. To determine the cellular relevance of this observation, we utilized the ANA-l murine macrophage model of endotoxin (LPS)-mediated NO synthesis. In selected instances, the NO synthase inhibitor, l-arginine methyl ester (L-NAME; 100 μM), or the NO donor, S-nitroso-N-acetylcysteine (SNAC; 100 μM), was added. In contrast to that of LPS cells, nuclear extracts from LPS+ L-NAME cells demonstrated increased NF-κB DNA binding on gel shift analysis. Addition of SNAC to LPS+ L-NAME cells restored binding to a level equivalent to that of LPS cells. Spectrophotometric analysis of NF-κB p50 immunoprecipitates demonstrated S-NO bonds exclusively in LPS cells; these p50 protein isolates retained the same DNA binding characteristics as that of the nuclear extracts. Transfection assays utilizing NF-κB-dependent promoter-reporter constructs demonstrated increased activity in LPS+ L-NAME cells compared with LPS cells; nuclear run-on assays confirmed increased transcription of the corresponding genes. These results suggest that LPS-mediated NO synthesis is associated with S-nitrosylation of NF-κB p50 and inhibition of NF-κB-dependent DNA binding, promoter activity, and gene transcription. We conclude that NO can regulate gene transcription by S-nitrosylation of NF-κB.