Angiotensin II (Ang II) is the major effector peptide of the renin angiotensin system that induces inflammatory gene expression through the nuclear factor-kappaB (NF-kappaB) transcription factor. Activation of latent cytoplasmic NF-kappaB is controlled by distinct pathways, the best known being the canonical pathway controlling IkappaB kinase activation. Interestingly, Ang II only weakly activates the canonical pathway. Although basal nucleocytoplasmic RelA shuttling is required for Ang II stimulation, changes in RelA translocation do not account for its transcriptional effect. Instead, Ang II rapidly induced RelA phosphorylation at Ser residue 536, and complex formation with the Ser(536) kinase known as the NF-kappaB-inducing kinase (NIK)/MEKK14. The requirement of NIK in Ang II-inducible transcription was shown by expressing a dominant-negative NIK or small interfering RNA (siRNA)-mediated knockdown; both inhibited Ang II-induced transcription. Conversely, constitutively active NIK potently induced RelA transactivation activity. Consistent with its actions independent of the canonical pathway, NIK induces the activity of the RelA transactivation domains -1 and -2 in constitutively nuclear GAL4-RelA fusion proteins that do not bind IkappaBalpha. Ang II induces NIK activity, phosphorylation of its endogenous IkappaB kinase alpha substrate, and induction of nuclear NF-kappaB2 (p52) processing. NIK down-regulation prevents Ang II-induced phospho-Ser(536) RelA formation, indicating that it is essential for RelA activation. The Ang II pathway further involves the RhoA small GTP-binding protein because RhoA inhibition blocks Ang II-induced transcriptional activity and formation of phospho-Ser(536) RelA formation. Finally, we demonstrate that Ang II infusion in vivo rapidly induces phospho-Ser(536) RelA formation and activation of the NF-kappaB-dependent IL-6 gene. These data indicate that Ang II induces NF-kappaB-dependent transcription through an alternative pathway, being largely independent of IkappaB proteolysis, but mediated by the small GTPases Rac/RhoA, required for NIK.RelA complex formation and inducible Ser(536) RelA phosphorylation.