Glutamate is thought to be a major excitatory neurotransmitter in the central nervous system. To study the glutamate receptor and its regulation under carefully controlled conditions, the specific binding of [3H]glutamate was characterized in washed membranes isolated from a neuroblastoma X retina hybrid cell line, N18-RE-105. [3H]Glutamate bound in a saturable and reversible fashion with an apparent dissociation constant, KD, of 650 nM and a maximum binding capacity, Bmax, of 16 pmol/mg of protein. Pharmacologic characterization of the site indicates that it closely resembles the Na+-independent binding site for glutamate found on brain membranes and thought to be an excitatory amino acid neurotransmitter receptor. Thus, while kainate, N-methyl-DL-aspartate, and nonamino acid ligands did not displace [3H]glutamate, quisqualate and ibotenate were potent inhibitors of specific binding. Furthermore, this binding site is regulated by ions in a manner which resembles that described in the hippocampus (Baudry, M., and Lynch, G. (1979) Nature (Lond.) 282, 748-750). Calcium (10 mM) increased the number of binding sites 2.6-fold with no change in receptor-ligand affinity. Lanthanum (1 mM) was the only other cation added which enhanced (3-fold) the binding of [3H]glutamate. Monovalent cations resulted in a decrease in the number of glutamate binding sites. Incubation of membranes in the presence of chloride ions caused a marked increased in [3H] glutamate binding, an effect which was synergistic with that of calcium incubation. Thus, N18-RE-105 cells possess a binding site for [3H]glutamate pharmacologically similar to an excitatory neurotransmitter binding site in brain and which exhibits regulatory properties resembling those previously described in hippocampal membranes, providing an excellent model for mechanistic studies.