This study investigated the in vitro (40 MHz) and in vivo (30 MHz) feasibility of intravascular ultrasound to document the influence of atherosclerotic lesions on the typical 3-layered appearance of muscular arteries. The in vitro images of 39 arteries were compared with the corresponding histologic sections. Media and lesions thickness were measured at the areas of minimal and maximal lesions thickness. The median media thickness was 0.8 mm in the absence of a lesion, decreasing to 0.3 mm in the area of maximal atherosclerosis. The ultrasonic data correlated closely with histologic measurements (0.6 and 0.3 mm, respectively). The in vivo study was performed in 29 patients under-going coronary or peripheral vascular procedures. A total of 150 still-frames were selected for quantitative analysis. The median media thickness was 0.6 mm in the absence of a lesion, decreasing to 0.1 mm in the area with maximal atherosclerosis. This study revealed that intravascular ultrasound imaging accurately determines that media thickness of muscular arteries is inversely related to lesion thickness. In vitro data, verified with histology, can be translated to humans in vivo.