Transient receptor potential vanilloid subtype 1 (TRPV1) channels are essential nociceptive integrators in primary afferent neurons. These nonselective cation channels are inhibited by local anesthetic compounds through an undefined mechanism. Here, we show that lidocaine inhibits TRPV1 channels expressed in Xenopus laevis oocytes, whereas the neutral local anesthetic, benzocaine, does not, suggesting that a titratable amine is required for high-affinity inhibition. Consistent with this possibility, extracellular tetraethylammonium (TEA) and tetramethylammonium application produces potent, voltage-dependent pore block. Alanine substitutions at Phe649 and Glu648, residues in the putative TRPV1 pore region, significantly abrogated the concentration-dependent TEA inhibition. The results suggest that large cations, shown previously to enter cells through activated transient receptor potential channels, can also act as channel blockers.