Transient receptor potential vanilloid type 1 (TRPV1) can be activated by multiple chemical and physical stimuli such as capsaicin, anandamide, protons, and heat. Capsaicin interacts with the binding pocket constituted by transmembrane regions 3 and 4, whereas protons act through residues in the prepore loop of TRPV1. Here, we report on characterization of polyclonal and monoclonal antibodies to the prepore loop of TRPV1. A rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) acted as a full antagonist of proton activation (IC(50) values for pH 5 and 5.5 were 364.68 +/- 29.78 and 28.31 +/- 6.30 nM, respectively) and as a partial antagonist of capsaicin, heat, and pH 6 potentiated chemical ligand (anandamide and capsaicin) activation (50-79% inhibition). Ab-156H antagonism of TRPV1 is not affected by the conformation of the capsaicin-binding pocket because it is equally potent at wild-type (capsaicin-sensitive) rat TRPV1 and its T550I mutant (capsaicin-insensitive). With the goal of generating monoclonal antagonist antibodies to the prepore region of human TRPV1, we used a recently developed rabbit immunization protocol. Although rabbit polyclonal antiserum blocked human TRPV1 activation, rabbit monoclonal antibodies (identified on the basis of selective binding to Chinese hamster ovary cells expressing human TRPV1) did not block activation by either capsaicin or protons. Thus, rabbit polyclonal antibodies against rat and human TRPV1 prepore region seem to partially lock or stabilize the channel in the closed state, whereas rabbit anti-human TRPV1 monoclonal antibodies bind to the prepore region but do not lock or stabilize the channel conformation.