In this study, we expressed rat vanilloid receptor 1 (VR1) in various heterologous expression systems using different VR1-encoding vectors, and examined how the VR1 agonists capsaicin and resiniferatoxin affected intracellular calcium. Our results clearly show that the magnitude and kinetics of response as well as the extent of tachyphylaxis differ markedly between systems. Using green fluorescent protein-tagged VR1, we show that much of the VR1 is localized to intracellular membranes. Consistent with this localization, VR1 agonists are able to liberate calcium from intracellular stores in the absence of extracellular calcium. As with other parameters of response, the three expression systems differ in the degree to which, in the absence of extracellular calcium, capsaicin and resiniferatoxin can liberate calcium from the intracellular stores. Our findings emphasize the influence of the expression system on characteristics of the response of VR1 to its ligands and the need for caution in extrapolating such results to other settings.