We hypothesized that in hyperglycemia activation of polyol pathway, via increased production of sorbitol by the aldose reductase, contribute to the development of endothelial dysfunction. Thus the role of polyol pathway on vasomotor function in isolated rat skeletal muscle arterioles (~150 μm, at 80 mmHg) was investigated. Compared to controls (5 mM glucose, CG), high glucose treatment (HG, 25 mM glucose for 60 min) reduced flow (0–40 μL/min)- and sodium nitroprusside (SNP, 10−9–10−6 M)-induced dilations (flow: CG max: 39 ±2 μm, HG max: 15 ±1μm; SNP: control max: 41 ±2μm, HG max: 33 ±2μm), whereas adenosine (10−7–10−4 M)-induced dilations remained intact (CG max.: 42 ±1μm, HG max: 42 ±2μm). Presence of the aldose reductase inhibitor (ARI, zopolrestat, 10 μM) did not affect responses of CG arterioles, but mitigated HG-induced decreases in flow-induced dilations (HG + ARI max: 27 ±2μm), and also ameliorated impaired SNP-induced dilations (HG + ARI max: 38 ±1μm) of HG arterioles. Also, compared to control there was increased superoxide production, indicated by ethidium bromide fluorescence, in HG treated femoral arteries, which was reduced by ARI. Thus we suggest that, increased activation of polyol pathway via increasing oxidative stress could contribute to the development of hyperglycemia-induced arteriolar dysfunction. Supported by AHA NE Aff. 0555897T and Hungarian NSRF/OTKA -T48376, HSC/ETT 364/2006 and Zsigmond Diabetes Found: 711-Dr.Koller/84289.