Objective: The overall goal of this project was to examine the interactions of hyperglycemia and loss of ovarian hormones on the artery wall in a type I diabetic mouse model. Methods and Results: Intact or ovariectomized (ovariectomy; OVX) female BALB/C mice were fed a high-cholesterol diet. Half the animals were treated with steptozotocin to induce insulin-deficient diabetes mellitus (D), generating 4 treatment groups: control, intact (CI); control, ovariectomized (COVX); diabetic, intact (DI); diabetic, ovariectomized (DOVX). We examined arterial structure and function and found: 1) Diabetes and ovariectomy additively increased endothelial layer permeability. 2) Arterial stiffening was increased in the diabetic and ovariectomy groups (DOVX). 3) Ovariectomy and diabetes (DOVX) synergistically increased atheroma formation. 4) Ultrastructural evaluation revealed that the basal lamina was often multilayered and formed convoluted aggregates separating endothelium from the internal elastic lamina in diabetic, but not control arteries or arteries from ovariectomized mice. Endothelium overlying these regions formed thin cytoplasmic extensions between these aggregates and was often separated from the basal lamina by electron lucent spaces. Conclusions: Our studies showed that diabetes and loss of ovarian function have additive and synergistic effects to worsen arterial pathophysiology by disrupting the arterial endothelial layer with increased permeability and increased atheroma formation.