A variety of polyamine spider and wasp toxins are known to block N-methyl-D-aspartate receptor channels and recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the edited glutamate receptor (GluR)2 subunit. Recently, inward rectification of GluR2-lacking AMPA receptors was shown to be caused by voltage-dependent block by intracellular spermine. Here we demonstrate that, when applied extracellularly, the endogenous polyamines spermine and spermidine, as well as monoacylated spermine analogs and the polyamine toxins ageltoxin-489 and philanthotoxin-433, exerted a use-dependent and weakly voltage-dependent block of AMPA receptors that lack the edited GluR2 subunit, when the recombinant receptors were expressed in Xenopus oocytes. External spermine and polyamine toxins were also effective blockers of AMPA receptor mutants that did not not show inwardly rectifying kainate responses but had high calcium permeability. The polyamines and polyamine toxins also markedly reduced inwardly rectifying currents of native AMPA receptors expressed by a class of hippocampal interneurons in rat CA3 stratum radiatum that appear not to express the GluR2 subunit. In contrast, polyamines had little or no effect on the linear or outwardly rectifying kainate responses of other interneurons or CA3 pyramidal cells in which GluR2 mRNA was routinely detected. Together with previous reports, these data suggest that endogenous polyamines may bind to GluR2-lacking AMPA receptors at two or more distinct sites, one near the cytoplasmic side of the pore and the other nearer the outer side of the pore.