H1 horizontal cells of goldfish retina probably are GABAergic and receive synaptic excitation from red cone photoreceptors in the dark. This study was designed to detect efflux of [3H]GABA from H1 cells by a physiological stimulus in order to obtain information regarding the identity of the red cone transmitter and obtain information on the role of dopaminergic interplexiform cells (DA-IPCs), the other synaptic input to H1 cells. Efflux of [3H]GABA was studied by biochemical analysis of perfused isolated retinas. Retinas were incubated in dim red light for 15 min in 0.72 microM [3H]GABA, rinsed for 30 min in red light and subjected to darkness under a variety of conditions. Radioactivity in the perfusate was determined by liquid scintillation spectroscopy. The findings are: 1. both L-glutamate and L-aspartate cause a dose-dependent efflux of [3H]GABA from H1 cells, 2. inclusion of 3.2 mM D-aspartate in the perfusion medium potentiates L-glutamate and totally inhibits L-aspartate, 3. retinas perfused in the standard Ringer do not show increased [3H]GABA efflux when placed in the dark, 4. when 3.2 mM D-aspartate is in the perfusion medium, there is significant dark-induced [3H]GABA efflux which is reduced with light onset, 5. 100 microM dopamine inhibits the dark-induced efflux of [3H]GABA. These results show that efflux of [3H]GABA from H1 cells can be detected under physiological conditions strongly suggesting that the H1 cell is GABAergic and, in addition, is subject to antagonistic inputs from red cones and DA-IPCs. Furthermore, since D-aspartate potentiates L-glutamate and inhibits L-aspartate, and is required for the detection of dark-induced efflux of [3H]GABA, it is unlikely that the transmitter for red cones is L-aspartate but more likely is L-glutamate.