Hyperglycemia increases glucose metabolism via the polyol pathway, which results in elevations of intracellular sorbitol concentration. Thus we hypothesized that elevated level of sorbitol contributes to the development of hyperglycemia-induced dysfunction of microvessels. In isolated, pressurized (80mmHg) rat gracilis muscle arterioles (~150µm), high glucose treatment (HG, 25mM)-induced reduction in flow dependent dilation (from max.:39 ±2 % to 15 +1 %), which was significantly mitigated by an aldose reductase inhibitor, zopolrestat (max.:27±2 %). Increasing doses of sorbitol (10^-10-10^-4 M) elicited dose-dependent constrictions (max.:22±3%), which was abolished by endothelium removal, a prostaglandin H₂/ thromboxane A₂ (PGH₂/TXA₂) receptor (TP) antagonist, SQ-29548 or superoxide dismutase (SOD) plus catalase (CAT). Incubation of arterioles with sorbitol (10-7 M) reduced flow dependent dilations (from max.: 39±2 to 20±1.5 %), which was not further affected by inhibition of NOS by L-NAME, but was prevented by SOD plus CAT and mitigated by SQ-29548. The NO donor, sodium nitroprusside-induced (10^-9-10^-6 M) dilations were also decreased in a SQ-29548 and SOD plus CAT reversible manner, whereas adenosine dilations were not affected by sorbitol exposure. Sorbitol significantly increased arterial superoxide production detected by lucigenin-enhanced chemiluminescense, which was inhibited by SOD plus CAT. Sorbitol treatment also increased arterial formation of 3-nitrotyrosine. We suggest that hyperglycemia by elevating intracellular sorbitol induces oxidative stress, which interferes with NO bioavailability and promotes PGH₂/TXA₂ release both of which affect regulation of vasomotor responses of arterioles. Thus increased activity of polyol pathway may contribute to the development of microvascular dysfunction in diabetes mellitus.