Tamoxifen and 17beta-estradiol are capable of up-regulating the expression of some genes and down-regulate the expression of others simultaneously in the same cell. In addition, tamoxifen shows distinct transcriptional activities in different target tissues. To elucidate whether these events are determined by differences in the recruitment of co-regulators by activated estrogen receptor-alpha (ER-alpha) at target promoters, we applied chromatin immunoprecipitation (ChIP) with promoter microarray hybridisation in breast cancer T47D cells and identified 904 ER-alpha targets genome-wide. On a selection of newly identified targets, we show that 17beta-estradiol and tamoxifen stimulated up- or down-regulation of transcription correlates with the selective recruitment of co-activators or co-repressors, respectively. This is shown for both breast (T47D) and endometrial carcinoma cells (ECC1). Moreover, differential co-regulator recruitment also explains that tamoxifen regulates a number of genes in opposite direction in breast and endometrial cancer cells. Over-expression of co-activator SRC-1 or co-repressor SMRT is sufficient to alter the transcriptional action of tamoxifen on a number of targets. Our findings support the notion that recruitment of co-regulator at target gene promoters and their expression levels determine the effect of ER-alpha on gene expression to a large extent.