1. The current study examined the hypothesis that endothelial production of hydrogen peroxide (H2O2) mediates relaxations to acetylcholine (ACh) in aorta and small mesenteric arteries (SMA) from mice. 2. Relaxations to ACh (0.01-10 microM) and H2O2 (0.1-1000 microM) were produced in aorta and SMA isolated from wild-type C57BL/6 mice and type II diabetic mice (db/db). In SMA, relaxations to ACh were produced in the presence of N omega-nitro-L-arginine methyl ester (100 microM) and indomethacin (Indo, 10 microM). 3. 1-H[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one (10 microM) significantly reduced ACh-induced relaxations in SMA, abolished responses in aorta, but had no effect on relaxations induced by H2O2. Catalase (2500 U ml-1) abolished responses to H2O2, but did not alter relaxations to ACh in the SMA and only caused a small rightward shift in responses to ACh in the aorta. 4. ACh-, but not H2O2-, mediated relaxations were significantly reduced by tetraethylammonium (10 mM), the combination of apamin (1 microM) and charybdotoxin (100 nM), and 25 mm potassium chloride (KCl). Higher KCl (60 mM) abolished relaxations to both ACh and H2O2. Polyethylene glycolated superoxide dismutase (100 U ml-1), the catalase inhibitor 3-amino-1,2,4-triazole (3-AT, 50 mM) and treatment with the copper chelator diethyldithiolcarbamate (3 mM) did not affect relaxations to ACh. 5. H2O2-induced relaxations were endothelium-independent and were not affected by ethylene diamine tetraacetic acid (EDTA 0.067 mM), 4-aminopyridine (1 mM), ouabain (100 microM) and barium (30 microM), 3-AT or Indo. 6. Although the data from this study show that H2O2 dilates vessels, they do not support the notion that H2O2 mediates endothelium-dependent relaxations to ACh in either aorta or SMA from mice.