Normal human left ventricular architecture comprises a highly aligned array of cardiac myofibers whose orientation depends on transmural location. This study was designed to determine whether measurement of integrated backscatter could be used detect the progressive transmural shift of myofiber alignment that occurs from epicardium to endocardium in human ventricular wall segments. Integrated backscatter was measured at 32 transmural levels in seven cylindrical biopsy specimens (1.4 cm diam) sampled from normal regions of six explanted fixed human hearts by insonification of samples at 180 independent angles in 2 degrees steps around their entire circumference with a 5-MHz broadband piezoelectric transducer. Histologic analysis was performed to determine fiber orientation. Integrated backscatter varied approximately as a sinusoidal function of the angle of insonification at each transmural level. Greater integrated backscatter was observed for insonification perpendicular as compared with parallel to fibers (difference = 14.5 +/- 0.6 dB). Ultrasonic analysis revealed a progressive transmural shift in fiber orientation of approximately 9.2 +/- 0.7 degrees/mm of tissue. Histologic analysis revealed a concordant shift in fiber orientation of 7.9 +/- 0.8 degrees/mm of tissue. Thus, human myocardium manifests anisotropy of ultrasonic scattering that may be useful for characterization of the intramural fiber alignment and overall three-dimensional organization of cardiac myofibers.