Most excitatory synaptic transmissions in the central nervous system are mediated by the neurotransmitter glutamate. Binding of glutamate released from the presynaptic membrane causes glutamate receptors in the postsynaptic membrane to open, which results in a transient depolarization of the postsynaptic membrane. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) subtype of glutamate receptors is responsible for the majority of excitatory postsynaptic currents and is thought to play a central role in synaptic plasticity. Because modulation of glutamate receptors is believed to be involved in the basic mechanism underlying information storage in the brain, the molecular architecture of native AMPA receptors (AMPA-Rs) is of great interest. Previously, we have shown that AMPA-Rs purified from the brain are tightly associated with members of the stargazin/TARP (transmembrane AMPA receptor regulatory protein) family of membrane proteins [Nakagawa et al., Nature 433 (2005), pp. 545-549]. Here, we present a three-dimensional (3D) density map of the hetero-tetrameric AMPA-R without associated stargazin/TARP proteins as determined by cryo-negative stain single-particle electron microscopy. In the absence of stargazin/TARP proteins, the density representing the transmembrane region of the AMPA-R particles is substantially smaller, corroborating our previous analysis that was based solely on projection images.