AMPA (alpha-amino-3-hydroxy-5-methyl-4-isooxazole) receptors (AMPARs) are glutamate-gated ion channels that play central roles in the mammalian brain, mediating fast excitatory synaptic transmission and underlying several forms of synaptic plasticity. Two subtypes of AMPARs are primarily coexpressed at excitatory synapses in adult animals (GluR1/2 and GluR2/3 receptors). Efforts to distinguish between the potentially distinct roles these receptor subtypes play at synapses have been hampered by the absence of a subtype-selective AMPAR antagonist. Here we show that the polyamine philanthotoxin-7,4 (PhTx-74) is a subtype-selective AMPAR antagonist, inhibiting GluR1/2, but not GluR2/3 receptors, use-dependently. The molecular basis for the observed selectivity and a highly efficient synthesis of this remarkable toxin are also presented. PhTx-74 provides the first a means of directly monitoring subtype-specific changes in the composition of AMPARs at native synapses, including during hippocampal long-term potentiation (LTP), a form of synaptic plasticity thought to share processes related to learning and memory.