Phosphorylation of histone H3 protein at serine 10 is an important step in chromatin remodeling during transcriptional transactivation. IkappaB kinase-alpha (IKK-alpha) and Mitogen- and Stress-activated protein Kinases 1 and 2 (MSK1/2) have been shown to play key roles in the transcriptional regulation of immediate early genes such as c-fos. Interestingly, IKK-alpha and MSK1/2 have also been implicated as histone H3-Ser10 kinases. In this work, we have shown that MSK1/2 are required for epidermal growth factor (EGF)-induced, but not tumor necrosis factor-induced, histone H3-Ser10 phosphorylation, both globally and at specific promoters. Consistent with this, MSK1/2 are required for optimal immediate early c-fos transcription in response to EGF potentially through control of both H3-Ser10 and promoter-associated cAMP-response element-binding protein phosphorylation. Furthermore, MSK1/2 control EGF-induced IkappaB alpha promoter H3-Ser10 phosphorylation in the absence of elevated transcription. These studies demonstrate the existence of pathway-specific mechanisms to control histone H3-Ser10 phosphorylation and gene expression.