The intravitreal administration of ACh agonists (eserine, carbachol, oxotremorine) or that of ACh muscarinic antagonists (scopolamine, atropine) provoked a reduction of the On-Off ganglion cell discharges. The agonists depressed the Off discharges more than the On discharges, while the ACh muscarinic antagonists depressed the On- more than the Off discharges. These drugs did not modify the ganglion cell receptive field area; thus, the muscarinic cholinergic system seems not to be involved in the spatial organization of the On-Off ganglion cells, but rather seems to play an important part in the separation of On and Off information channels. ACh nicotinic antagonists [hexamethonium, D-tubocurarine (D-TC), alpha-bungarotoxin (alpha-BGTX)] provoked an increase of the receptive field area of On-Off ganglion cells, this enlargement being due to the suppression of the inhibition normally exerted by the surround upon the centre of the field. Moreover D-TC and alpha-BGTX, but but hexamethonium, increased the number of ganglion cell discharges. These data are analogous to those obtained after administration of GABA antagonists and show that through nicotinic receptors, ACh seems to be involved in the spatial organization of the On-Off ganglion cell.