Estrogens exert a key biological role in mammary gland epithelial cells and promote breast carcinogenesis and tumor progression. We recently identified a new large set of estrogen responsive genes from breast cancer (BC) cells by DNA microarray analysis of the gene expression profiles induced by 17beta-estradiol in ZR-75.1 and MCF-7 cells. The purpose of the present study was to test whether the expression pattern of hormone regulated genes from this set identifies estrogen receptor (ERalpha) positive, hormone responsive BC cells. To this aim, we carried out in silico metanalysis of ERalpha positive and ERalpha negative human BC cell line transcriptomes, focusing on two sets of 171 and 218 estrogen responsive genes, respectively. Results show that estrogen dependent gene activity in hormone responsive BC cells is significantly different from that of non-responsive cells and, alone, allows to discriminate these two cellular phenotypes. Indeed, we have identified 61 genes whose expression profile specifically marks ERalpha positive BC cells, suggesting that this gene set may be exploited for phenotypic characterization of breast tumors. This possibility was tested with data obtained by gene expression profiling of BC surgical samples, where the ERalpha positive phenotypes were highlighted by the expression profile of a subset of 27 such hormone responsive genes and four additional BC marker genes, not including ERs. These results provide direct evidence that the expression pattern of a limited number of estrogen responsive genes can be exploited to assess the estrogen signaling status of BC cells both in vitro and ex-vivo.