1. Reactive oxygen species (ROS) cause vascular complications and impair vasodilation in diabetes mellitus. Large-conductance Ca(2+)-activated potassium channels (BK(Ca)) modulate vascular tone and play an important negative feedback role in vasoconstriction. In the present study, we tested the hypothesis that ROS regulate the function of BK(Ca) in diabetic cerebral artery smooth muscle cells. 2. Diabetes was induced in male BALB/c mice by injection of streptozotocin (STZ; 180 mg/kg, i.p., dissolved in sterile saline). Control and diabetic mice were treated with 12.7 micromol/L rotenone, an inhibitor of the mitochondrial electron transport chain complex I, or placebo every other day for 5 weeks. The whole-cell patch clamp-technique and functional vasomotor methods were used to record BK(Ca) currents and myogenic tone of cerebral artery smooth muscle cells. 3. In the diabetic group, there was a significant decrease in spontaneous transient outward currents in cerebral artery smooth muscle cells compared with control. Although the currents were only moderately increased in rotenone-treated diabetic mice, they remained significantly lower than in the control group. Furthermore, the macroscopic BK(Ca) currents that were decreased in diabetic mice were partially recovered in rotenone-treated diabetic mice (P < 0.05 vs untreated diabetic group). 4. The posterior cerebral artery from diabetic mice had a significantly higher myogenic tone than the control group, but this impaired contraction was partially reversed in the rotenone-treated diabetic group (P < 0.05 vs untreated diabetic group). 5. The H(2)O(2) concentration was significantly increased in cerebral arteries from diabetic mice compared with control. This increase in H(2)O(2) was significantly blunted by rotenone treatment. 6. In conclusion, rotenone partially reverses the decreased macroscopic BK(Ca) currents in STZ-induced Type 1 diabetic mice and this reversal of BK(Ca) currents may be related to the inhibitory effects of rotenone on H(2)O(2) production. Reactive oxygen species, particularly H(2)O(2), are important regulators of BK(Ca) channels and myogenic tone in diabetic cerebral artery.