The cholinergic amacrine cells of the rabbit retina can be labeled with [3H]choline and the activity of the cholinergic population monitored by following the release of [3H]acetylcholine. It has been proposed that L-homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [3H]acetylcholine. In magnesium-free medium, D-homocysteate was slightly more potent than the L-isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to L-homocysteate. 2-Amino-7-phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked L-homocysteate evoked responses. 6,7-Dinitroquinoxaline-2,3-dione, a potent kainate antagonist, preferentially blocked D-homocysteate-evoked responses. Therefore, in the rabbit retina, L-homocysteate is an NMDA-preferring agonist, whereas D-homocysteate is a kainate-preferring agonist. In addition, we found that L-homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA-receptor activation. However, the low potency of L-homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of L-homocysteate at the bipolar-to-cholinergic amacrine cell synapse.