L-Glutamate elicits an inwardly rectifying current at hyperpolarized potentials in isolated retinal cones of the tiger salamander, as measured under whole-cell patch clamp. Evidence presented in this article supports the notion that cones possess a high-affinity glutamate transporter. This glutamate-elicited current shows no desensitization over a period of several minutes, and has an affinity (Km) of 10 microM. The inward current is mimicked by the amino acids L-aspartate, D-aspartate, L-cysteate, and to a lesser extent D-glutamate. It is neither blocked by the glutamate receptor antagonists kynurenic acid (1 mM), 6-cyano-7-nitroquinoxaline-2,3-dione (100 microM), or 2-amino-5-phosphonovalerate (100 microM), nor elicited by the glutamate receptor agonists (100 microM each) kainate, quisqualate, NMDA, or 2-amino-4-phosphonobutyrate. The glutamate-elicited current was reduced by the glutamate transport blockers dihydrokainate (DHKA), DL-threo-beta-hydroxyaspartate (beta HA), and L-trans-pyrrolidine-2,4-dicarboxylic acid. When glutamate was present on both sides of the membrane, the blockers reduced both uptake and release; the blocker-sensitive current as a function of membrane potential represents the transport current-voltage relation (I-V), and the reversal potential of the I-V represents the transporter equilibrium potential. This potential was a function of the equilibrium potential for glutamate. DHKA and beta HA depolarized horizontal cells in a retinal slice, and abolished their light responses, suggesting that in the absence of glutamate transport, glutamate concentrations in the cleft rise to a level that saturates the postsynaptic receptors. The high capacity of the cone glutamate transporter is well suited for the rapid removal of glutamate from the synaptic cleft required for the signaling of a light onset to postsynaptic cells.