Intracellular recordings were obtained from horizontal and bipolar cells of the superfused, isolated retina-eyecup of the rabbit. The putative neurotransmitters aspartate, glutamate, and several analogues were added to the superfusate while the membrane potential and light-responsiveness of the retinal neurons were monitored. Both L-aspartate and L-glutamate mimicked the actions of the endogenous photoreceptor transmitter on horizontal cells, on-bipolar cells, and off-bipolar cells. At applied concentrations of 2.5-20 mM, the actions of L-aspartate and L-glutamate were indistinguishable. D-aspartate potentiated the effects of both L-aspartate and L-glutamate on horizontal cells. This suggests that active uptake systems for these amino acids exist in the outer plexiform layer (OPL) of the rabbit retina. The glutamate analogue kainate produced effects similar to those of aspartate and glutamate on second-order neurons, but at concentrations lower by over two orders of magnitude. The glutamate analogue quisqualate had effects similar to kainate but with much less potency. The aspartate analogue n-methyl DL-aspartate (NMDLA) antagonized the effects of the photoreceptor transmitter on horizontal and off-bipolar cells. This action of NMDLA was only observed at low concentrations (50 microM). In addition, NMDLA could block the effects of exogenously applied kainate. The NMDLA had no clear effects on on-bipolar cells. The glutamate analogue 2-amino-4-phosphonobutyrate reversibly blocked the responses of on-bipolar cells but had no effect on either horizontal or off-bipolar cell responses. This suggests that on-bipolar cells possess a unique synaptic receptor. The aspartate analogue 2-amino-3-phosphonoproprionate did not show this selectivity, suggesting that this unique receptor is a glutamate-preferring receptor. The antagonists alpha-methyl glutamate, alpha-amino adipate, and glutamate diethyl ester all showed only a weak ability to antagonize the actions of the photoreceptor transmitter on second-order neurons. The results of this study indicate that glutamate or a glutamate-like substance is the likely transmitter of rods and cones in the rabbit retina. A comparison of the present findings with those previously obtained in lower vertebrate retinas suggests that the basic pharmacological design of the OPL of all vertebrate retinas is very similar.