Background. A number of studies suggest that premenopausal diabetic women loose the gender based cardiovascular protection. However, there is insufficient evidence to establish the timeline or the mechanism(s) underlying the loss of premenopausal female specific cardiovascular protection in presence of hyperglycemia. Objectives. The objectives of our study were to investigate (1) if there is a gender based difference in the relaxation response of rat and rabbit aorta after acute exposure to high glucose (HG) concentration, and (2) the potential role of protein kinase C (PKC) isoforms (-α,-β,-δ), superoxide and Rho kinase in the HG-induced vascular dysfunction. Methods. Endothelium-dependent vasodilation (EDV) to acetylcholine (ACh, 10 -8 to 10 -5 M) was determined before and after 3 h treatment with Krebs' solution containing HG (46 mM) in aortic rings pre-contracted with phenylephrine (2 μM) taken from female and male rats (Sprague-Dawley) and rabbits (New Zealand White). Similar experiments were generated in the presence of 9 nM Ro-32-0432, a selective PKC-α inhibitor, 1 μM LY379196, a selective PKC-β inhibitor, 6 μM Rottlerin, a selective PKC-δ inhibitor, 25 μM MnTMPyP, a superoxide scavenger or 1 μM Fasudil, a Rho kinase inhibitor. Furthermore, the effect of acute estrogen administration on hyperglycemia-induced endothelial dysfunction was evaluated in male and female rabbits. Lastly, mRNA and protein expression of PKC isoforms were measured by RT-PCR and Western blotting, respectively. Results. We demonstrated that (1) a 3 h incubation with elevated level of glucose impairs ACh responses only in the female rat aortic rings but not in males. A similar exposure to HG impairs relaxation to a greater extend in female than male rabbit aorta, (2) inhibition of PKC-β but not Rho kinase prevents the HG-induced impairment of endothelium-dependent relaxation of female rat and rabbit aorta, (3) superoxide scavenging significantly corrected the vascular impairment caused by hyperglycemia in female rat and rabbits, and (4) PKC-β expression is significantly higher in female rat and rabbit aorta than in their male counterparts. Furthermore, acute 17β-estradiol aggravates HG-induced endothelial dysfunction in female rabbit aorta but not in male. Conclusion. This study reveals the predisposition of female rat and rabbit aorta to vascular injury under hyperglycemic conditions, possibly via activation of PKC-β and superoxide production. Furthermore, it suggests that under hyperglycemic conditions acute estrogen treatment is detrimental to endothelial function in female rabbit.