The role of endothelial calcium signaling in exercise-enhanced ACh-induced vasorelaxation was examined using male Wistar rats (8~10 wk old) that were divided into control and exercise groups. The exercised animals ran on a treadmill with progressive increments of speed until exhaustion. After decapitation, aortic rings were dissected and loaded with fura 2-AM. After being mounted on a tissue flow chamber, vessels were precontracted with phenylephrine, and ACh-induced endothelial calcium elevation and vasorelaxation were determined simultaneously under an epifluorescence microscope equipped with ratio imaging capability. Our results showed that 1) there was logarithmic correlation between endothelial calcium elevation and vasorelaxation; 2) acute exercise enhanced ACh-induced endothelial calcium elevation and vasorelaxation without altering their relationship; 3) pretreatment with N-nitro-L-arginine markedly reduced ACh-induced vasorelaxation in both groups but suppressed the calcium response only in the exercise group; and 4) the exercise effect on endothelial calcium elevation was abolished by Ca²⁺-free buffer or gadolinium. In conclusion, acute exercise increases ACh-induced vasorelaxation by increasing the endothelial calcium influx and the calcium-dependent nitric oxide release.