1. In outside-out patches excised from human embryonic kidney (HEK) 293 cells expressing Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) channels, currents activated by 1 ms glutamate pulses at negative membrane potentials facilitated during and following a repetitive (2 to 100 Hz) agonist application. The degree of facilitation depended on subunit type, membrane potential and stimulation frequency being antagonized by a slow recovery from desensitization. 2. Activity-dependent current facilitation occurred in Ca2+-permeable but not in Ca2+-impermeable AMPAR channels. Current facilitation, however, does not depend on Ca2+ flux. Rather it reflects a relief from the block of Ca2+-permeable AMPARs by intracellular polyamines since facilitation occurred only in the presence of polyamines and since facilitated currents had a nearly linear current-voltage relation (I-V). 3. Relief from polyamine block was use dependent and occurred mainly in open channels. The relief mechanism was determined primarily by membrane potential rather than by current flow. 4. In closed channels the degree of polyamine block was independent of membrane potential. The voltage dependence of the rate of relief from the block in open channels rather than the voltage dependence of the block underlies the inwardly rectifying shape of the I-V at negative potentials. 5. Currents through native Ca2+-permeable AMPAR channels in outside-out or nucleated patches from either hippocampal basket cells or a subtype of neocortical layer II nonpyramidal cells also showed facilitation. 6. It is concluded that a use-dependent relief from polyamine block during consecutive AMPAR channel openings underlies current facilitation. This polyamine-AMPAR interaction may represent a new activity-dependent postsynaptic mechanism for control of synaptic signalling.