The NF-kappaB family of transcription factors is an important component of stress-activated cytoprotective signal transduction pathways. Previous studies demonstrated that some activation mechanisms require phosphorylation, ubiquitination, and degradation of the inhibitor protein, IkappaBalpha. Herein, it is demonstrated that ionizing radiation in the therapeutic dose range stimulates NF-kappaB activity by a mechanism in which IkappaBalpha tyrosine 181 is nitrated as a consequence of constitutive NO* synthase activation, leading to dissociation of intact IkappaBalpha from NF-kappaB. This mechanism does not appear to require IkappaBalpha kinase-dependent phosphorylation or proteolytic degradation of IkappaBalpha. Tyrosine 181 is involved in several noncovalent interactions with the p50 subunit of NF-kappaB stabilizing the IkappaBalpha-NF-kappaB complex. Evaluation of hydropathic interactions of the IkappaBalpha-p50 complex on the basis of the crystal structure of the complex is consistent with nitration disrupting these interactions and dissociating the IkappaBalpha-NF-kappaB complex. Tyrosine nitration is not commonly studied in the context of signal transduction. However, these results indicate that tyrosine nitration is an important post-translational regulatory modification for NF-kappaB activation and possibly for other signaling molecules modulated by mild and transient oxidative and nitrosative stresses.