The purpose of this study was to validate intracoronary ultrasound imaging by correlation with histologic examination. In this in-vitro study of pressure-perfused human coronary arteries, 104 matching intracoronary ultrasound imaging images and histologic cross-sections from 12 hearts were compared to determine the diagnostic accuracy of 30 MHz commercially available intracoronary ultrasound imaging. For lipid deposits, sensitivity was 46% and specificity 97%. The smallest lipid deposit that was visualized measured 0.25 mm in axial diameter on histologic study. For calcific deposits, sensitivity was 77% and specificity 100%. The smallest calcific deposit that was visualized measured 0.25 mm in axial diameter on histologic examination. Atherosclerotic intimal thickening could not be distinguished qualitatively or quantitatively from nonatherosclerotic intimal thickening unless there were localized deposits of lipids or calcium. Intracoronary ultrasound imaging is accurate in detecting lipid and calcium deposits. Intimal thickening in intracoronary ultrasound imaging images does not prove the presence of atherosclerosis.