Abstract: Vanilloid receptor 1 (TRPV1), a membrane associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands. Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable 3H-RTX binding in membranes prepared from their dorsal root ganglia. Like it's mammalian orthologs, cloned rabbit TRPV1 (oTRPV1) is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (M547 and T550) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, 3H-RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders. Vanilloid receptor 1 (TRPV1), a membrane associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands. Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable 3H-RTX binding in membranes prepared from their dorsal root ganglia. Like it's mammalian orthologs, cloned rabbit TRPV1 (oTRPV1) is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (M547 and T550) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, 3H-RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.