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In addition to endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium-derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H2O2) in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh).ACh (10−6, 10−5, and 10−4 M) induced a transient contraction of rat renal artery with intact endothelium in a concentration-related manner, but not in the artery with endothelium removed. In phenylephrine-precontracted renal arteries, ACh induced an endothelium-dependent relaxation response at lower concentrations (10−8-10−6 M), and a relaxation followed by a contraction at higher concentrations (10−5 M). Inhibition of nitric oxide synthase by Nω-nitro-L-arginine (10−4 M) enhanced the EDC to ACh.Catalase (1000 U ml−1) reduced the EDC to ACh. H2O2 (10−6, 10−5, and 10−4 M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium-independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh-induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17-octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H2 receptors (SQ 29548) and thromboxane A2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H2O2.In isolated endothelial cells, ACh (10−5 M) induced a transient H2O2 production detected with a fluorescence dye sensitive to H2O2 (2′,7′-dichlorofluorescein diacetate). The peak concentration of H2O2 was 5.1 × 10−4 M at 3 min and was prevented by catalase.Taken together, these results show that ACh triggers H2O2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H2O2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H2O2 is most likely the EDCF in rat renal artery in response to ACh stimulation.