Divalent cations Mg 2+ and Ba 2+ selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg 2+ potentiates channel activation only from the extracellular side; on the intracellular side, Mg 2+ inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1-S2 linker, the S3-S4 linker, and the pore turret are all required for Mg 2+ potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg 2+ -induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg 2+ also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg 2+ precedes channel activation, suggesting that Mg 2+ -induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.