The capsaicin receptor (TRPV1) is a nonselective cation channel that is activated in nociceptors by several painful stimuli, and hence TRPV1 antagonists could represent a novel class of analgesic compounds. Resiniferatoxin (RTX), a potent agonist of TRPV1, and iodoresiniferatoxin (I-RTX), a potent antagonist of TRPV1, both bind with higher affinity to the rat TRPV1 (rTRPV1) than the human (hTRPV1) isoform. To identify the structural features responsible for this difference in affinity, [(3)H]RTX binding to chimeras between hTRPV1 and rTRPV1 was characterized. The "sensor" region within the transmembrane domain (S1-S4) was found to determine [(3)H]RTX binding affinity. All 16 different residues in this region were systematically substituted in hTRPV1 with rTRPV1 residues. A single mutation in the S4 membrane domain of hTRPV1, L547M, caused a 30-fold increase in [(3)H]RTX affinity whereas the inverse mutation in rTRPV1, M547L, caused a 30-fold decrease in affinity for [(3)H]RTX, and several other agonists and antagonists were similarly affected by these mutations. TRPV1 channels with mutations at position 547 were expressed in oocytes, and the relative response to RTX followed a pattern similar to that seen with [(3)H]RTX binding. These data suggest a model where Met-547 in the S4 domain of TRPV1 forms a binding pocket with Tyr-511 in the S3 domain. This model places RTX near the sensor domain thought to move during the gating process and should help to guide further work designed to understand the gating mechanisms of TRPV1 channels based on comparisons between the agonist RTX and the related competitive antagonist I-RTX.