RSKB, a 90-kDa ribosomal S6 protein kinase family (RSK) member with two complete catalytic domains connected by a linker, is activated through p38- and ERK-mitogen-activated protein kinases. The N-terminal kinases of RSKs phosphorylate substrates; activation requires phosphorylation of linker and C-terminal kinase sites. Unlike other RSKs, the activation loop phosphorylation sites of both catalytic domains of RSKB, Ser(196) and Thr(568), were required for activity. RSKB activation depended on phosphorylation of linker Ser(343) and Ser(360) and associated with phosphorylation of nonconserved Ser(347), but Ser(347)-deficient RSKB retained partial activity. The known protein kinase A and protein kinase C inhibitors, H89 and Ro31-8220, blocked RSKB activity. Treatment of HeLa cells with tumor necrosis factor, epidermal growth factor, phorbol 12-myristate 13-acetate, and ionomycin but not with insulin resulted in strong activation of endogenous RSKB. High RSKB activity and Ser(347)/Ser(360) phosphorylation persisted for 3 h in tumor necrosis factor-treated cells, in contrast to the short bursts of p38, ERK, and RSK1-3 activities. In conclusion, a variety of stimuli induced phosphorylation and activation of RSKB through both p38 and ERK pathways; the persistence of activation indicated that RSKB selectively escaped cell mechanisms causing rapid deactivation of upstream p38 and ERK and other RSKs.