Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.