We obtained intracellular recordings from transient, On-Off amacrine and ganglion cells of the turtle retina. We tested the ability of neurotransmitter agonists and antagonists to modify the responses to light stimuli. The metabotropic glutamate agonist, 2-amino-phosphonobutyric acid (APB), selectively blocked On responses, whereas the amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor antagonist, GYKI, blocked both On and Off responses. Although GYKI appeared to block excitation completely, suggesting an absence of N-methyl-d-aspartate (NMDA)-mediated responses, it was found that in the presence of ionotropic gamma-aminobutyric acid (GABA) blockers, the excitatory postsynaptic potential (EPSP) was prolonged. The late component of the EPSP was blocked by the NMDA antagonist, D-2-amino-5-phosphopentanoic acid (D-AP5). Picrotoxin (PTX) and bicuculline (BCC) induced a mean hyperpolarization of -6.4 mV, suggesting a direct effect of GABA on transient amacrine and ganglion cells, since antagonism of a GABA-mediated inhibition of release of glutamate by bipolars would depolarize third-order neurons. The acetylcholine agonist, carbachol, or the nicotinic agonist, epibatidine, depolarized all On-Off neurons. This action was blocked by d-tubocurarine. Cholinergic inputs to On-Off neurons increase their excitability without altering the pattern of light responsiveness.