Procedures for the determination of the spatial distribution and the dynamic changes of intracellular Ca²⁺ signaling in single intact living cells are described using laser scanning con focal microscopy (LSCM) and the highly fluorescent Ca²⁺ -sensitive dye, fluo-3/AM. The duration, dye concentration, and requirement for Pluronic are determined empirically for the loading conditions. It is especially important for the proper intracellular dye concentration to provide adequate signal strength for detection while not to disturb normal intracellular physiology. For example, in C57BL/6J macrophages, it was found that cells cultured on glass cover slips were incubated for 1 h at 37℃ with 6 μmol/L fluo-3/AM, resulting in excellent LSCM imaging of intracellular Ca²⁺.The similar results have been successfully obtained by this method for other types of cells such as vascular smooth muscle cells, Chinese hamstar oocytes. It may provide an available, visual experimental approach for investigation into the dynamic changes of Ca²⁺ signal and their relationship to transmembrane Ca²⁺ gradient in living cells under the physiological and pathological conditions.