Abstract: Objective To explore the kinetic changes of calcium in rat smooth muscle cells and establish a method for quantification of intracellular calcium. Methods Rat mesenteric arteriolar smooth muscle cells (ASMCs) were isolated and the kinetic changes of calcium were measured using highly sensitive Ca²⁺ fluorescent probe indo-1 with laser scanning confocal microscopy (LSCM). The dissociation constant values (Kd) of the fluorescent probe indo-1 was measured at 37 ℃, and according to the conversion formula from fluorescence intensity to concentration, the concentration of Ca²⁺ was calculated. Results Analysis of the fluorescent images using LSCM showed that[Ca²⁺]_i in the ASMCs were significantly elevated in response to stimulation with dexamethasone, and spontaneous calcium waves as well as intracellular calcium overloading were observed occasionally. Conclusion Fluorescence quantification with LSCM is applicable for detecting the kinetic changes of intracellular[Ca²⁺]_i.