Little is known about the interaction between diabetes and sex in vasculature. This study was designed to investigate whether there were sex differences in rat aortic endothelial function in diabetes, and to examine the potential roles of superoxide and nitric oxide (NO) in this sex-specific effect. Two diabetic animal models were used: streptozotocin (STZ)-induced type 1 diabetic rats (at early and intermediate stages of disease) and Zucker type 2 diabetic fatty (ZDF) rats. Endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) was measured in aortic rings pre-contracted with phenylephrine (PE) before and after pretreatment with MnTmPYP (10 mM), a superoxide scavenger, or apocynin (100 μM), a NADPH oxidase (Nox) inhibitor. Constrictor response curves (CRC) to PE (10 -8 to 10 -5 M) were also generated before and after pretreatment with L-NAME (200 μM), an endothelial nitric oxide synthase (eNOS) inhibitor, in the presence of indomethacin. In addition, the level of Nox (a potent source of superoxide) and eNOS mRNA expression were determined using real-time RT-PCR. STZ-induced diabetes impaired EDV to ACh to a greater extent in female than male aortae both at early and intermediate stage of disease (1- and 8- week, respectively). Incubation of aortic rings with L-NAME potentiated PE responses in all groups, but aortae from control females showed a greater potentiation of the PE response after NOS inhibition compared with others. STZ-diabetes reduced the extent of PE potentiation after L-NAME and the aortic eNOS mRNA expression in females to the same levels as seen in males. In addition, pre-incubation with MnTMPyP enhanced sensitivity to ACh only in diabetic females one week after STZ induction. Similarly, the levels of Nox1 mRNA expression were enhanced in STZ-induced diabetic females. Type 2 diabetes significantly impaired EDV in aortic rings from females; however, it potentiated the relaxation in male rats. Moreover, type 2 diabetes enhanced the extent of PE potentiation after blocking NOS with L-NAME in females. Pre-incubation of aortic rings with apocynin increased EDV only in diabetic female group. Accordingly, the levels of Nox1, Nox4 and eNOS mRNA expression were substantially enhanced in aorta of female ZDF rats compared to those in lean animals. In a conclusion, our data suggest that an elevation of superoxide and alteration of NO production may in part contribute to the predisposition of the female aorta to injury in diabetes.