Previous studies showed that arteriolar tone is enhanced in type 2 diabetes mellitus (T2-DM) due to increased level of constrictor prostaglandins. We hypothesized that in mice with T2-DM hydrogen peroxide (H₂O₂) contributes to the increased synthesis of constrictor prostaglandins, hence enhanced basal tone in skeletal muscle arterioles. Isolated, pressurized gracilis muscle arterioles (~100 µm in diameter) of mice with T2-DM (C57BL/KsJ-db^-/db^-) exhibited greater basal tone to increases in intraluminal pressure (20-120 mmHg), than control vessels (at 80 mmHg, control: 25±5%, db/db: 34±4%, p<0.05), which was reduced back to control level by catalase (db/db: 24±4%). Correspondingly, in carotid arteries of db/db mice the level of dichlorofluoresceine-detectable and catalase-sensitive H₂O₂ was significantly greater. In control arterioles, exogenous H2O2 (0.1-100 µmol/L) elicited dilations (max: 58±10%), whereas in arterioles of db/db mice H₂O₂ caused constrictions (-28±8%), which were converted to dilations (max: 16±5%) by the thromboxane A₂/prostaglandin H₂ (TP) receptor antagonist, SQ29548. In addition, arteriolar constrictions to the TP receptor agonist, U46619 (0.1-10 µmol/L) were not different between the two groups of vessels. Endothelium denudation did not significantly affect basal tone and H₂O₂-induced arteriolar responses in either control or db/db mice. Also, in arterioles of db/db mice, but not in controls, 3-nitrotyrosine staining was detected in the endothelial layer of vessels. Thus, we propose that in mice with T2-DM, arteriolar production of H₂O₂ is enhanced, which leads to increased synthesis of constrictor prostaglandins, TXA₂/PGH₂ to enhance the basal arteriolar tone. These alterations may contribute to disturbed regulation of skeletal muscle blood flow in type 2 diabetes mellitus.