Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ER) and , estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPCR) GPR30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical estrogen receptor (ER), the regulation of GPR30 expression by 17-estradiol, the selective GPR30 ligand G-1, Insulin Growth Factor-1 (IGF-1) and Epidermal Growth Factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an AP-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction which was strongly recruited to the AP-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGFR-MAPK transduction pathway stimulated a regulatory loop which subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGFR-MAPK signaling provides new insight into the well-known estrogen and EGF cross-talk which, as largely reported, contributes to breast cancer progression. On the basis of our results the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen even in ER-negative breast tumor cells.