Deregulation of the HER2 oncogene occurs in 30% of human breast cancers and correlates with poor prognosis and increased propensity for metastasis. Since the molecular basis of HER2 overexpression in human cancers is not known, we sought to determine whether chromatin remodeling pathways are involved in the regulation of HER2 expression. We report that compared with breast cancer cells expressing a low level of HER2, HER2-overexpressing breast cancer cells contained significantly higher levels of acetylated and phosphorylated histone H3, and acetylated histone H4 associated with the HER2 promoter. Decreased recruitment of histone deacetylases in the promoter is also noted in the HER2-overexpressing cell. The association of acetylated histone H4 with HER2 gene chromatin and HER2 expression in breast cancer cells was upregulated by an inhibitor of histone deacetylases. Treatment with histone deacetylase inhibitor also reduced the association of histone deacetylase-1 and -2 with the HER2 promoter. In addition, the tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid stimulated the association of phosphorylated histone H3 on serine 10 with the HER2 promoter and also stimulated HER2 expression. These findings identify histone acetylation and histone phosphorylation as novel regulatory modifications that target HER2 gene chromatin, and suggest that elevated levels of these chromatin-relaxing components in the vicinity of the HER2 gene promoter may constitute an important non-genomic mechanism of HER2 overexpression in human breast cancer.