Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins I-B. Previously we reported that the extracellular signal-regulated kinase (ERK) is required for interleukin-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, interleukin-1 activated ERK and induced degradation of both I-B and I-B, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)-1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an I-B/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with interleukin-1 decreased I-B in the RSK1/I-B/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced I-B decrease without influencing ether ERK phosphorylation or the earlier I-B degradation. By using recombinant wild type and mutant I-B proteins, both active ERK2 and RSK1 were found to directly phosphorylate I-B, but only active RSK1 phosphorylated I-B on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates I-B and contributes to the persistent activation of NF-B by IL-1.