l-Glutamic acid (l-Glu) and other excitatory amino acids and amino acid analogs enhanced [35S]thiocyanate (SCN-) uptake in isolated-resealed synaptic membrane vesicles. The SCN- uptake was used as a measure of membrane depolarization to evaluate the characteristics of functional excitatory amino acid receptors in the synaptic membranes. N-Methyl-d-aspartate (NMDA) and l-Glu produced additive effects on SCN- accumulation indicating the presence of distinct l-Glu and NMDA receptors. On the other hand, kainic acid (KA) and l-Glu shared either common receptor sites or ion channels. The effects of antagonists on NMDA, l-Glu, and KA stimulation of SCN- influx were consistent with previously reported electrophysiologic observations in intact neurons.