Histone H3 phosphorylation is a downstream response to activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. This modification is thought to have a role in chromatin remodeling and in the initiation of gene transcription. In MCF-7 breast cancer cells, we observed that phosphorylated histone H3 (phospho-H3) at Ser(10) but not Ser(28) increased with phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) treatment. Although phosphorylated extracellular signal-regulated kinase 1/2 levels in these cells cultured under estradiol deplete and replete conditions displayed no change, a significant induction was observed after TPA treatment. Furthermore, whereas both estradiol and TPA increased trefoil factor 1 (TFF1) mRNA levels in these cells, only TPA-induced and not estradiol-induced TFF1 expression was inhibited by the H3 kinase mitogen and stress activated protein kinase (MSK) inhibitor H89 and MAPK kinase inhibitor UO126, showing the involvement of the Ras/MAPK following TPA induction. Mutation of the activator protein 1 (AP-1) binding site abrogated the TPA-induced transcriptional response of the luciferase reporter gene under the control of the TFF1 promoter, showing the requirement for the AP-1 site. In chromatin immunoprecipitation assays, estradiol treatment resulted in the association of the estrogen receptor-alpha (ERalpha) and acetylated H3 with the TFF1 promoter. The levels of phospho-H3 and MSK1 associated with the TFF1 promoter were moderately increased. In the presence of TPA, whereas ERalpha was not bound to the promoter, a strong association of acetylated and/or phospho-H3, MSK1, and c-Jun was observed. These results show that although both stimuli lead to TFF1 gene activation, estradiol and TPA exert their effects on TFF1 gene expression by different mechanisms.