Glutamate and neurosteroids are known to exist in retinal ganglion cells (RGC). Therefore, patch clamp studies using the whole-cell recording method were performed to determine whether or not ionotropic glutamate receptor subtypes, i.e., N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors, were present on RGC obtained by the magnetic cell sorter (MACS) method and cultures. In addition, the effects of 20-hydroxyecdysone (20-HE), a neurosteroid, on inward currents induced by NMDA, AMPA and kainate were examined at a holding potential of -60 mV. The current-voltage relationship for NMDA in the presence of glycine and Mg2+-free, as well as those for AMPA and kainate were linear, with a reversal potential of around 0 mV. NMDA-induced currents were blocked by MK-801, while both AMPA- and kainate-induced currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Application of 20-HE in the bath resulted in significant inhibitions on NMDA-, AMPA- and kainate-induced currents. Thus, NMDA, AMPA and kainate receptors were confirmed to exist on MACS-separated cultured RGC. Moreover, 20-HE inhibited NMDA receptor-mediated currents most prominently and AMPA- and kainate-mediated currents moderately, suggesting that neurosteroids may be playing a role in modulating glutamate-mediated transmission in RGC, and 20-HE might be useful for preventing glutamate neurotoxicity.