Estrogen receptor alpha (ERalpha)-negative breast cancer cells display an aggressive phenotype. We previously showed that adenoviral expression of ERalpha in ER-negative breast cancer cells leads to an estrogen-dependent down-regulation of the proliferation, which could be of interest to control the growth of such cells. In this study, we observed an increase in protein levels of p21 and p27 cyclin-dependent kinase inhibitors, whereas pRb phosphorylation is strongly decreased. Flow cytometry experiments showed a slower transit of cells in G1 (hormone-independent), a hormone-induced accelerated transit through S phase and a possible arrest in G2/M phase. In addition, ERalpha-expressing cells were undergoing apoptosis. By using cDNA macroarrays, we identified a novel collection of genes regulated by liganded ERalpha potentially regulating cell cycle, apoptosis, cell signalling, stress response and DNA repair.