Local hyperthermia has been the subject of much research because of its great potential for therapeutic and clinic applications. It has been long recognized that a major factor, which affects tissue temperature elevation and heterogeneity during hyperthermia, is the augmentation of blood flow concomitant with the heating. The heat-induced change in local blood flow can be attributed to sympathetically mediated re-distribution of cardiac output and change in local flow resistance resulting from thermally stimulated regulation in diameters of arterioles. It has been found that the vascular endothelium significantly affects the dynamic response of the vessel diameter to thermal stimuli. Endothelial cells play key regulatory roles by producing several potent vasoactive agents and regulating coagulation states, i.e. endothelium derived relaxing factors (EDRFs). Most endothelial functions depend to various extents on changes in intracellular calcium concentration [Ca2+]i. A new approach to studying vascular thermo-regulation during hyperthermia has been developed in this research to quantitatively measure the dynamic response of vascular endothelial Ca2+ to temperature elevations using confocal fluorescence ratio imaging. The cell membrane permeable fluorescence dye Fura-2/AM esters were loaded into the vascular endothelial cells and ratio imaging of the fluorescent endothelial cell were taken under the excitation of 334 and 380nm wavelengths. The signal intensities were calibrated with the endothelial calcium ion concentration ([Ca2+]i) and temperatures ranged from 37°C to 44°C. This calibration will provide a means to quantitatively measure the vascular endothelial [Ca2+]i transients in in vivo tissue when subjected to temperature elevations from 38°C to 44°C, and thus to further understand the role of endothelium in thermally induced vascular regulation under hyperthermic conditions in the near future.