Polyamines are important endogenous regulators of ion channels and are known to modulate inflammation and nociception. Here we investigated effects of polyamines on the capsaicin receptor TRPV1, a major ion channel expressed in nociceptive sensory afferents. Extracellular spermine, spermidine, and putrescine directly activated TRPV1 in a charge-dependent manner, both in heterologous expression systems and sensory neurons. The threshold for activation by spermine was approximately 500 microm at room temperature. At lower concentrations, spermine enhanced capsaicin-evoked currents with an EC50 of approximately 5 microm. Further, polyamines freely permeated TRPV1 (estimated relative permeabilities compared with Na+ were between 3 and 16), and spermine reduced the single channel conductance from 96 to 49 pS. Experiments with TRPV1 mutants identified extracellular acidic residues critical for polyamine regulation. Neutralization of aspartate 646 (D646N) abolished direct activation by spermine, whereas neutralization of this same aspartate (D646N) or glutamate 648 (E648A) inhibited spermine-induced sensitization. These data show that polyamines, by virtue of their cationic charge, can regulate the activity of TRPV1. Extracellular polyamines are present in considerable concentrations in the gastrointestinal tract and at synapses, and these levels increase during inflammation and cancer. Therefore, polyamine regulation of TRPV1 in these tissues may be relevant to a variety of physiological and pathophysiological states.