In this study, we investigated the effects of estradiol on the proliferation of neonatal keratinocytes, the expression of estrogen receptor isoforms, and the signaling mechanisms by which estradiol mediates cell growth. We demonstrate that estradiol binds neonatal keratinocytes with high affinity (Kd=5.2nM) and limited capacity (Bmax of 14.2fmol/mg of protein), confirming the presence of estrogen binding sites. Using specific antibodies, we demonstrate that keratinocytes express both estrogen receptor (ER)-alpha and ER-beta. At physiological concentrations, estradiol up-regulates the level of ER-alpha receptors in keratinocytes and induces keratinocyte proliferation. The proliferative effect of estradiol requires the availability of functional estrogen receptors, as it is abrogated by anti-estrogen administration. Estradiol effect on keratinocyte proliferation is most likely mediated in part by activation of a nongenomic, membrane-associated, signaling pathway involving activation of the extracellular signal regulated kinases 1 and 2 and in part by the genomic signaling pathway through activation of nuclear receptors.