The vanilloid receptor 1 (VR1) is a ligand-gated, nonselective cation channel important for the sensory processing of painful stimuli. Activation of VR1 leads to increases in intracellular concentrations of calcium and sodium. Prolonged activation of VR1 in mammalian expression systems leads to cell death. The mechanism of VR1-mediated toxicity may have relevance to pathophysiological processes that can occur in neurons. Therefore, we have evaluated the relative contributions of intracellular calcium and sodium changes to VR1-mediated toxicity in human embryonic kidney 293 cells stably transfected with the human VR1 channel. The data demonstrate that VR1 receptor agonists capsaicin and resiniferatoxin lead to a sustained increase in intracellular calcium and sodium in a concentration-dependent manner, followed by cell death. Pretreatment with VR1 receptor antagonists capsazepine or ruthenium red block both the calcium and sodium responses to agonists, and block agonist-induced cell death in a concentration-dependent manner. However, addition of antagonists several minutes after agonists selectively reverses the agonist-induced increase in intracellular calcium, but does not reverse the elevated intracellular sodium concentration. Nonetheless, antagonists retain protective efficacy against capsaicin toxicity when added several minutes after capsaicin, conditions in which the cells still manifest elevated intracellular sodium, but not elevated intracellular calcium. In addition, a transient VR1-mediated increase in intracellular calcium that returns to baseline within minutes, induced by a rapid drop in pH, from pH 7.5 to pH 6.3, also does not lead to cell death. Collectively, these data demonstrate that the most important intracellular ionic change for mediating VR1-dependent toxicity is a sustained increase of calcium.