AIMS: The density of vasa vasorum within atherosclerotic plaque correlates with histologic features of plaque vulnerability in post-mortem studies. Imaging methods to non-invasively detect vasa vasorum are limited. We hypothesized that contrast ultrasound (CUS) can quantify vasa vasorum during atherosclerosis progression. METHODS AND RESULTS: New Zealand white rabbits received a high-fat diet for 3 weeks, and bilateral femoral artery stenosis was induced by balloon injury. Contrast ultrasound femoral imaging was performed at baseline and 2, 4, and 6 weeks post injury to quantify adventitial videointensity. At each imaging time point 10 vessels were sectioned and stained with haematoxylin and eosin and von-Willebrand factor. Adventitial vasa vasorum density was quantified by counting the number of stained microvessels and their total cross-sectional area. Plaque size (per cent lumen area) progressed over time (P < 0.001), as did adventitial vasa vasorum density (P < 0.001). Plateau peak videointensity also progressed, demonstrating a strong linear correlation with histologic vasa vasorum density (P < 0.001). Receiver operating characteristic analysis indicated that a three-fold increase in median adventitial videointensity had a sensitivity of 100% and specificity of 88% for predicting abnormal neovascularization. CONCLUSION: We have histologically validated that CUS quantifies the development of adventitial vasa vasorum associated with atherosclerosis progression. This imaging technique has the potential for characterizing prognostically significant plaque features.