Protein and messenger RNA levels of the AMPA-type glutamate receptor subunits 1-3 are high in many brain regions, but it is not known how much of the glutamate receptor protein is expressed on the surface of neurons in the form of functional receptors. To provide insight into this matter, western blot immunoreactivities for glutamate receptors 1 and 2/3, as well as binding of the specific ligand [3H]AMPA, were quantified following three independent treatments modifying surface receptors in intact primary hippocampal cultures: (i) proteolysis of surface receptors by chymotrypsin, (ii) cross-linking of surface receptors with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate, and (iii) biotinylation of surface receptors with the membrane-impermeant reagent sulfosuccinimidyl-2(biotinamido)ethyl-1,3-dithiopropionate. All three of these methods demonstrated that 60-70% of total glutamate receptor subunit 1 protein and 40-50% of total glutamate receptor 2/3 protein are expressed on the surface of hippocampal neurons. Parallel studies revealed that 52% of total [3H]AMPA binding sites could be precipitated with avidin beads following biotinylation of intact cultures, providing an estimate of [3H]AMPA binding site surface expression in accord with the estimates of the surface expression of glutamate receptor subunits 1-3. Experiments examining the surface expression of 32P-labeled glutamate receptor subunit 1 demonstrated that approximately 65% of the phosphorylated form of the subunit is located in the plasma membrane, an estimate similar to the that derived via western blot for the entire glutamate receptor subunit 1 population in the same samples. Moreover, no significant change in the surface expression profile of the glutamate receptor subunits 1-3 was observed following stimulatory treatments known to increase glutamate receptor phosphorylation. These data indicate that slightly more than half of the AMPA receptors in cultured hippocampal neurons are located in the plasma membrane, and that AMPA receptor surface expression is not rapidly altered by glutamate receptor phosphorylation.