The glutamate analogue 2-amino-4-phosphonobutyric acid (AP4) is a potent antagonist at several synapses where an excitatory amino acid appears to be the neurotransmitter. Previous studies identified a Cl-/Ca2+ dependent [3H]glutamate binding site in synaptic plasma membrane (SPM) preparations that was also labeled by [3H]AP4 and exhibited a pharmacology similar to the AP4 receptor. This report examines the pharmacological specificity in both biochemical and electrophysiological preparations in greater detail. Several compounds are identified which readily interact with the apparent binding site in membranes, but neither mimic nor inhibit the action of AP4 in electrophysiological studies. The rate of dissociation of [3H]AP4 from SPMs is shown to increase in the presence of added AP4 and increasing the osmolarity in the SPM binding assay decreases the level of observed [3H]AP4 binding. These findings indicate both a heterogeneous population of binding sites and the occurrence of transport. It is concluded that much of the AP4 binding observed in SPM preparations is to a site other than the AP4 receptor. The results provide a further pharmacological description of AP4 receptors which should facilitate the identification of the receptor in biochemical preparations.