17-α-ethinyl estradiol (EE) is a semi-synthetic hormone and the main component of combined contraceptive pills and drugs with therapeutic indications such as hormone replacement therapy. In Europe and the USA, approximately sixty million women use oral contraceptives, and it is estimated that nearly two million unintended pregnancies occur each year due to medication errors, potentially exposing the embryo to EE during the first weeks of development. The hippocampus is a glutamatergic brain area expressing hormone receptors and hormones influence synaptogenesis. The development of glutamatergic synapses is regulated in a precise time window by the GluN2B/GluN2A switch of the NMDAR subunits. This study investigates the EE effect on the expression and distribution of NMDAR and AMPAR. Primary rat hippocampal neurons are exposed to EE concentrations with different exposure schemes to cover the whole maturation period or the GluN2B/GluN2A switch relevant time window. Then, neurons are analyzed by Western blot and RT-PCR for NMDAR and AMPAR subunits in the homogenate and at the postsynaptic site. Results obtained after exposure to 200 nM EE suggest that the developmental program of the glutamatergic system is altered by EE at concentrations relevant to human exposure. The effect depends on the exposure time window and involves altered expression or translocation to the synaptic spines of selected subunits of NMDAR and AMPAR. Moreover, morphological analysis by confocal microscopy in GFP-transfected neurons showed a decrease in mature mushroom-shaped spines suggesting possible consequent functional effects which are now under investigation with live calcium imaging experiments.