Objectives: To investigate the functional role of hypercholesterolemia associated myocardial neovascularization in early atherosclerosis using the anti-angiogenic thalidomide. Background: Experimental atherosclerosis is characterized by myocardial neovascularization, associated with a decrease in myocardial perfusion response to challenge, coronary endothelial dysfunction and high oxidative stress. However, the functional significance of these neovessels is not known. Methods: three groups of pigs (n=6 each) were studied after 12 weeks of normal or hypercholesterolemic diet without (HC) or with thalidomide (HC+Thal). Myocardial perfusion and permeability were assessed at baseline and in response to cardiac challenge, using electron beam computed tomography and coronary endothelial function was assessed using organ chambers. Myocardial samples were scanned ex-vivo with a 3D micro-CT scanner, and the spatial density of the myocardial microvessels was quantified. Growth factors and oxidative stress were measured in the myocardial tissue. Results: Myocardial perfusion response to adenosine and dobutamine was blunted in both HC and HC+Thal compared to normal (p<0.05 HC and HC+Thal vs. normal) as was the coronary endothelial function. Myocardial permeability response to adenosine was increased in both HC and HC+Thal compared to normal (p<0.05 HC and HC+Thal vs. normal, and HC+Thal vs. HC). The microvascular density was increased in HC pigs compared to normal, but normalized in HC+Thal (p<0.001 HC vs. normal and HC+Thal). HC+Thal showed decreased expression of Flk-1 and b-FGF, but increased expression of VEGF, compared to normal and HC. Oxidative stress was increased in both HC and HC+Thal compared to normal. Conclusion: Chronic administration of thalidomide attenuates myocardial neovascularization in experimental HC without affecting myocardial perfusion response to stimulation. This suggests that the myocardial neovascularization may not contribute to the attenuated myocardial perfusion response in hypercholesterolemia.