The activation of NF-κB has been shown to be regulated by multiple phosphorylations of IκBs and the NF-κB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-κB stimulants including TNF-α in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-α- but not IL-1β-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IκB phosphorylation, inhibited the TNF-α-induced phosphorylation in vivo. In addition, overexpression of TGF-β-activated kinase 1 (TAK1), IKKα and IKKβ stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-α-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKα and IKKβ blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IκBα and p65, which further support the idea that both IκB and NF-κB are substrates for the IKK complex in the activation of NF-κB.