Two subtypes of the estrogen receptor (ER), ERalpha and ERbeta, mediate the actions of estrogens, and although 70% of human breast cancers express ERbeta along with ERalpha, little is known about the possible comodulatory effects of these two ERs. To investigate this, we have used adenoviral gene delivery to produce human breast cancer (MCF-7) cells expressing different levels of ERbeta, along with their endogenous ERalpha, and have examined the effects of ERbeta and receptor occupancy, using ER subtype selective ligands, on genome-wide gene expression by microarray and pathway network analysis. ERbeta had diverse effects on gene expression, enhancing or counteracting ERalpha regulation for distinct subsets of estrogen target genes. Strikingly, ERbeta in the absence of estradiol (E2), elicited the stimulation or suppression of many genes that were normally only regulated by ERalpha with E2. In addition, ERbeta plus E2 elicited the expression of a unique group of genes that were not regulated by ERalpha plus E2 alone. The expression of genes in many functional categories were modulated by ERbeta, with the greatest numbers associated with transcription factors and signal transduction pathways. Regulation of multiple components in the TGFbeta and semaphorin pathways, and of genes controlling cell cycle progression and apoptosis, may contribute to the suppression of cell proliferation observed with ERbeta. Our observations suggest that the relative levels of ERbeta and ERalpha in breast cancers are likely to impact cell proliferation and the activities of diverse signaling pathways and their response to ER ligands and endocrine therapies.