Regulator of G-protein signaling (RGS) proteins 2 (RGS2) and 4 (RGS4) play an important role in regulating G(i/o)- and G(q)-coupled receptors. In the present study, we investigated the possible impact of RGS2 and RGS4 on modulation of glycine currents of rat retinal ganglion cells (RGCs) mediated by the G(i/o)-coupled melatonin MT(2) receptor, using immunohistochemistry, Western blot analysis and whole-cell patch-clamp techniques. By immunofluorescence labeling the expression profiles of RGS2 and RGS4 proteins were basically similar. Both of them were widely expressed in the rat retina, particularly in the inner plexiform layer (IPL) and the ganglion cell layer (GCL). In addition, sparse signals of RGS2 and RGS4 were also detected in the inner nuclear layer (INL). Double immunofluorescence labeling further showed that all of RGCs retrogradely labeled expressed both RGS2 and RGS4. Western blot analysis confirmed the presence of RGS2 and RGS4 proteins in the rat retina. Intracellular dialysis of RGCs with the antibody against RGS2/RGS4 to block RGS2/RGS4 function gradually increased glycine current amplitudes of these cells. In the presence of the RGS2/RGS4 antibody melatonin-induced potentiation of glycine currents of RGCs was not observable. These results suggest that RGS2/RGS4 are coupled to melatonin receptor signaling in rat RGCs and these proteins may regulate the MT(2) receptor to change melatonin-induced modulation of glycine currents in rat RGCs.