The rapid induction of the c-fos gene correlates with phosphorylations of histone H3 and HMGN1 by mitogen- and stress-activated protein kinases. We have used a cell-free system to dissect the mechanism by which MSK1 phosphorylates histone H3 within the c-fos chromatin. Here, we show that the reconstituted c-fos chromatin presents a strong barrier to histone H3 phosphorylation by MSK1; however, the activators (serum response factor, Elk-1, cAMP-response element-binding protein (CREB), and ATF1) bound on their cognate sites recruit MSK1 to phosphorylate histone H3 at Ser-10 within the chromatin. This activator-dependent phosphorylation of histone H3 is enhanced by HMGN1 and occurs preferentially near the promoter region. Among the four activators, CREB plays a predominant role in MSK1-mediated phosphorylation of histone H3, and the phosphorylation of Ser-133 in CREB is essential for this process. Mutational analyses of MSK1 show that its N-terminal inhibition domain is critical for the kinase to phosphorylate chromatin-embedded histone H3 in a CREB-dependent manner, indicating the presence of an intricate regulatory network for MSK1-mediated phosphorylation of histone H3.