Transient receptor potential vanilloid-1 (TRPV1) ion channels are polymodal signal integrators of noxious stimuli including heat, vanilloids such as capsaicin, peptide toxins, acid, and inflammatory mediators. It is unknown whether activation of TRPV1 by different stimuli is achieved through the same structural mechanism or if different stimuli activate the channel through different structural mechanisms. Clinical trials using TRPV1 antagonists resulted in patients exhibiting hyperthermia, suggesting that TRPV1 plays a role in maintaining body temperature, and highlighting the need to ensure that therapeutics targeting the channel do not disrupt thermal homeostasis. Hence, knowledge of different structural mechanisms for channel activation would aid in the design of therapeutic agents targeting TRPV1. To address this, we have expressed a series of functional single-cysteine rat TRPV1 channels for spectroscopic analysis, with techniques such as electron paramagnetic resonance, double electron-electron resonance, and Förster resonance energy transfer spectroscopy. By probing several structural regions within TRPV1 we can determine which regions of the channels move during activation and whether those are the same for different noxious stimuli.