Ultrasound tissue characterization with measurement of backscatter has been employed in numerous experimental and clinical studies of cardiac pathology, yet the cellular components responsible for scattering from cardiac tissues have not been unequivocally identified. This laboratory has proposed a mathematical model for myocardial backscatter that postulates the fibrous extracellular matrix (ECM) as a significant determinant of backscatter. To demonstrate the importance of ECM, this group sought to determine whether measurements of backscatter from the isolated ECM could reproduce the known directional dependence, or anisotropy of backscatter, from intact cardiac tissues in vitro. Segments of left ventricular free wall from ten formalin fixed porcine hearts were insonified at 50 MHz, traversing the heart wall from endo- to epicardium to measure the anisotropy of myocardial backscatter, defined as the difference between peak (perpendicular to fibers) and trough (parallel to fibers) backscatter amplitude. The tissue segments were then treated with 10% NaOH to dissolve all of the cellular components, leaving only the intact ECM. Scanning electron micrographs (SEM) were obtained of tissue sections to reveal complete digestion of the cellular elements. The dimensions of the residual voids resulting from cell digestion were approximately the diameter of the intact myocytes (10-30 μm). These samples were reinsonified after seven days of treatment to compare the anisotropy of integrated backscatter. The magnitude of anisotropy of backscatter changed from 15.4±0.8 to 12.6±1.1 dB for intact as compared with digested specimens. Because digestion of the myocardium leaves only extracellular sources of ultrasonic scattering, and because the isolated ECM exhibits similar ultrasonic anisotropy as does the intact myocardium, it is concluded that there is a direct association between the ECM and the anisotropy of backscatter within intact tissue. Thus, it is suggested that ultrasonic tissue characterization represents a potentially clinically applicable method for delineating the structure and function of the ECM.