A theoretical model is described for application in ultrasonic tissue characterization using a calibrated 2-D spectrum analysis method. This model relates 2-D spectra computed from ultrasonic backscatter signals to intrinsic physical properties of tissue microstructures, e.g., size, shape, and acoustic impedance. The model is applicable to most clinical diagnostic ultrasound systems. Two experiments employing two types of tissue architectures, spherical and cylindrical scatterers, are conducted using ultrasound with center frequencies of 10 and 40 MHz, respectively. Measurements of a tissue-mimicking phantom with an internal suspension of microscopic glass beads are used to validate the theoretical model. Results from in vitro muscle fibers are presented to further elucidate the utility of 2-D spectrum analysis in ultrasonic tissue characterization.