The influences of frequency and spatial resolution on the anisotropic impedance estimation of cortical bone was investigated in the frequency range 25-100 MHz. A set of spherically focused transducers provided a spatial resolution in the range from 150 down to about 20 mum. Four embedded cortical bone samples (two male, two female, two donors aged <30 years, two donors aged >70 years) were cut with different orientations relative to the long axis of the femur (0-90 degrees ). From each section, impedance maps were acquired in the C-scan mode. Histogram evaluations showed a similar angular dependence with a characteristic off-axis maximum of the estimated impedance for all samples and frequencies. The impedance values obtained with the 25-MHz transducer were significantly lower than those obtained with the 50- and 100-MHz transducers. Morphological parameters of the macrostructure, for example, size and distribution of the haversian channels and the resulting porosity, were estimated from the high-resolution acoustic images. These structures appeared to have a significant influence on the measured properties of the bone matrix for the low-frequency and low-aperture measurements.