Calcific aortic valve sclerosis involves inflammatory processes and occurs preferentially on the aortic side of endothelialized valve leaflets. Although the endothelium is recognized to play critical roles in focal vascular sclerosis, the contributions of valvular endothelial phenotypes to aortic valve sclerosis and side-specific susceptibility to calcification are poorly understood. Using RNA amplification and cDNA microarrays, we identified 584 genes as differentially expressed in situ by the endothelium on the aortic side versus ventricular side of normal adult pig aortic valves. These differential transcriptional profiles, representative of the steady state in vivo, identify globally distinct endothelial phenotypes on opposite sides of the aortic valve. Several over-represented biological classifications with putative relevance to endothelial regulation of valvular homeostasis and aortic-side vulnerability to calcification were identified among the differentially expressed genes. Of note, multiple inhibitors of cardiovascular calcification were significantly less expressed by endothelium on the disease-prone aortic side of the valve, suggesting side-specific permissiveness to calcification. However, coexisting putative protective mechanisms were also expressed. Specifically, enhanced antioxidative gene expression and the lack of differential expression of proinflammatory molecules on the aortic side may protect against inflammation and lesion initiation in the normal valve. These data implicate the endothelium in regulating valvular calcification and suggest that spatial heterogeneity of valvular endothelial phenotypes may contribute to the focal susceptibility for lesion development.