It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-κB (p50−p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-κB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. Herein we show that cellular NF-κB activity is in fact regulated by S-nitrosylation. We observed that both S-nitrosocysteine and cytokine-activated NOS2 inhibited NF-κB in human respiratory cells or murine macrophages. This inhibition was reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity did not affect the TNFα-induced degradation of IκBα or the nuclear translocation of p65. Recapitulation of these conditions in vitro resulted in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect was reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 was detected in cells, and the level was modulated by TNFα. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-κB regulation.