Background: Changes in the structure of the arterial wall commence shortly after exposure to cardiovascular risk factors like hypercholesterolemia (HC), but may be difficult to detect. The ability to study vascular wall structure could be helpful to evaluate the instigating factors and pathomechanisms of atherosclerosis. This study tested the hypothesis that early morphological changes in coronary arteries of HC pigs can be detected using a novel x-ray contrast agent, osmium tetroxide (OsO₄), and 3D micro-computed tomography (CT). Methods: Two groups of pigs were studied after 12 weeks of normal or HC (2% cholesterol) diet. Hearts were harvested, coronary arteries were injected with 1% OsO₄ solution, and cardiac samples scanned by micro-CT (6µm section thickness). The layers of the epicardial coronary artery wall, early lesions, and perivascular OsO₄ accumulation were determined. The leakage of OsO₄ from myocardial microvessels was used to assess vascular permeability, which was correlated with immunoreactivity of vascular endothelial growth factor (VEGF) in corresponding histological cross sections. Results: OsO₄ enhanced the visualization of coronary arterial wall layers, and facilitated detection of early lesions in HC in longitudinal tomographic sections of vascular segments. Increased density of perivascular OsO₄ in HC was correlated with increased VEGF expression, and suggested increased microvascular permeability. Conclusion: The use of OsO₄ as a contrast agent in micro-CT allows 3D visualization of coronary arterial wall structure, early lesion formation, and changes in vascular permeability. Therefore, this technique can be a useful tool in atherosclerosis research.