It has been suggested that the NF-κB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-κB, the ability of NO-donor compounds to influence NF-κB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-κB p50 and p65 homodimers and of p50–p65 heterodimers. Inhibition of NF-κB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-κB p50 peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-κB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-κB-responsive genes.