13Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual 14 information, is a potentially better alternative to electrical stimulation widely used in current retinal 15 prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no retinal 16 stimulation study exploiting the inner retinal pathways exists. Here, we demonstrate the feasibility 17 of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the 18 retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that 19 controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas 20 elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON 21 RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally 22 injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way 23 for future development of a neurotransmitter-based subretinal prosthesis offering more 24 naturalistic vision and better visual acuity than electrical prostheses.25