Estrogen receptors (ERs) are a recognized prognostic factor and therapeutic target in breast cancer. The loss of ER expression relates to poor prognosis, poor clinical outcome and impairs the use of anti-estrogenic treatment. Histone deacetylase inhibitors are candidate drugs for cancer therapy. Among them, valproic acid (VPA) is a long used and safe anti-epileptic drug. We studied the biological consequences of the chromatin remodeling action of VPA in a normal human mammary epithelial cell line and in ERα-positive and ERα-negative breast cancer cell lines. In these cells and regardless of their ER status, VPA-induced cell differentiation, as shown by increased milk lipids production, decreased expression of the CD44 antigen and growth arrest in the G0-G1 phase of the cell cycle. These effects were accompanied by decreased Rb phosphorylation, hyperacetylation of the p21WAF1/CIP1 gene promoter and increased p21 protein expression. Only in breast cancer cells, cyclin B1 expression was decreased and the cells accumulated also in G2. ERα expression decreased in ERα-positive, increased in ERα-negative and was unchanged in normal mammary epithelial cells, as did the expression of progesterone receptor, a physiological ERα target. VPA decreased the expression of the invasiveness marker pS2 in ERα-positive breast cancer cells, but did not cause its re-expression in ERα-negative cells. Overall, these data suggest that in both ERα-positive and -negative malignant mammary epithelial cells VPA reprograms the cells to a more differentiated and "physiologic" phenotype that may improve the sensitivity to endocrine therapy and/or chemotherapy in breast cancer patients. © 2008 Elsevier Ltd. All rights reserved.