Epidemiological data suggest hyperglycemia abrogates the gender-based cardiovascular protection possibly associated with estrogens. This study was designed to investigate 1) whether rabbit aortic rings show any gender differences in the development of abnormal endothelium-dependent vasodilation (EDV) under acute hyperglycemic conditions, 2) the potential role of protein kinase C (PKC) isoforms and superoxide in acute hyperglycemia-induced vascular dysfunction, and 3) the effect of acute estrogen administration on hyperglycemia-induced endothelial dysfunction in male and female rabbits. EDV to acetylcholine was determined before and after 3 h treatment with high glucose (HG) in phenylephrine pre-contracted aortic rings from male and female New Zealand White rabbits. Similar experiments were conducted in the presence of inhibitors of PKC-, PKC-, PKC- or a superoxide scavenger. The effect of acute estrogen administration was evaluated in the presence and absence of HG. Finally, mRNA expression of PKC isoforms was measured by real-time PCR. We found that 1) 3 h incubation with HG impairs EDV to a greater extent in female than male aorta, 2) inhibition of PKC- or superoxide prevents HG-induced impairment of EDV in female aorta, 3) acute 17 -estradiol aggravates HG-induced endothelial dysfunction in female aorta but not in male, and 4) PKC- and PKC- expression are significantly higher in female than in male aorta. This study reveals the predisposition of female rabbit aorta to vascular injury under hyperglycemic conditions, possibly via activation of PKC- and superoxide production. Furthermore, it suggests that under hyperglycemic conditions acute estrogen treatment is detrimental to endothelial function in female rabbits.