The effects of D-O-phosphoserine (DOS) were examined on proximal neurons in the superfused mudpuppy retinal-eyecup preparation by measuring their synaptically evoked whole-cell currents with the use of patch-clamp electrodes. DOS reduced the light-evoked excitatory postsynaptic potentials (EPSPs) of amacrine and ganglion cells. This suppression was present even though the center responses of both ON- and OFF-bipolar cells were unaffected by DOS. When recordings were done under voltage-clamp conditions, DOS diminished the magnitude of light-evoked synaptic currents associated with a reduction in synaptic conductance. To determine which acidic amino acid receptor mediated the network-selective action of DOS, various glutamate agonists were tested against this excitatory amino acid receptor (EAAR) antagonist. DOS blocked the depolarizing effects of kainate (KA), but not those of N-methyl-D-aspartate (NMDA) or quisqualate (QQ). Thus DOS was a selective KA antagonist, and KA receptors appear to be the dominant EAAR subtype that mediates synaptic inputs into the inner retina of the mudpuppy.