Glutamate transporters in the tiger salamander retina were studied by autoradiographic and intracellular recording techniques. When the retina was incubated with 15 microM L-[3H]glutamate, photoreceptors and Muller cells were labeled, indicating that these cells had high-affinity glutamate uptake transporters. A much higher dose of glutamate than kainate was required in the bath to produce the same membrane depolarization in horizontal cells (HCs), and the time course of glutamate-induced depolarization was much slower than that of the kainate-induced depolarization. Since glutamate is a substrate of glutamate transporters whereas kainate is not, we attribute these differences to the buffering of extracellular glutamate by glutamate transporters in the retina. D-aspartate (D-asp) increased the efficacy of bath-applied glutamate. Dihydrokainate (DHKA) exerted little effect on glutamate efficacy when applied alone, but it increased glutamate efficacy in the presence of D-asp. These results are consistent with the notion that glutamate transporters in Muller cells are D-asp sensitive and those in photoreceptors are DHKA and D-asp sensitive. Application of DHKA (1-2 mM) did not affect the dark membrane potential or the light responses in rods and cones, but it depolarized the HC dark membrane potential and reduced the HC peak and tail light responses. Our results suggest that DHKA-sensitive glutamate transporters in photoreceptors regulate glutamate levels in rod and cone synaptic clefts. They modulate dark membrane potential and the relative rod cone inputs in retinal HCs.