A filtration binding assay using [3H]dichlorokynurenic acid to label the glycine binding site on the N-methyl-D-aspartic acid receptor has been evaluated on rat cortical membranes. This ligand binds to a single population of binding sites following mass action kinetics with a KD of 29 nM and a capacity of 5.73 pmol (mg protein)-1. The pharmacological specificity of the binding site is identical to that previously reported for this binding site using [3H]glycine as a radioligand. Agonists showed lower affinity and antagonists higher affinity when [3H]dichlorokynurenic acid was used compared with [3H]glycine. The higher affinity of [3H]dichlorokynurenic acid compared with [3H]glycine make it the more suitable compound with which to label the glycine site.