Gallstones (n = 20) were classified by dual energy Computer Tomography (CT) into three main classes: pure cholesterol stones (I), combination stones (II) and calcium stones (III). Further subclassification was possible by using morphological criteria. The acoustic measurements that were performed were measurements of the velocity of sound, the attenuation coefficient slope with frequency and intercept at 4.5 MHz, the attenuation coefficient slope per unit of time, the backscattering characteristics, and the appearance of B-mode echograms. The velocity of sound in calcium stones (c = 1695 +/- 107 m/s) was distinctly lower than in those containing some cholesterol (c > 2000 m/s). The attenuation coefficient slope ranged from 4.3 to 16.2 dB/cm MHz, the 4.5 MHz intercept from 21 to 66.2 dB/cm. The lowest values were found for the pure cholesterol stones (class IA), the highest values for subclass IIB (combination stones with shell). The attenuation coefficient slope per unit of time was distinctly lower (< 0.50 dB/microseconds MHz) for the cholesterol stones than for the combination and calcium stones (>0.64 dB/microseconds MHz). The backscattering spectrum was approximated by a straight line fit, and the slope for the cholesterol stones was lower than for the combination and calcium stones (<0.75 dB/MHz vs. >1.0 dB/MHz, respectively). The latter two parameters were assessed by in vivo applicable methods. The front echo level was found to be more than 5 dB higher for class IIB as compared to the other classes, while the spectral backscatter level at 4.5 MHz was considerably higher for both classes IIB and III. The B-mode echograms showed that a strong front echo in combination with a strong attenuation of the remaining echo signals was mainly found for stones of classes IIB and III. A significant difference between the group of stones that are suitable for lithotripsy and dissolution treatment (classes IA, IB and IIA) and the nontreatable calcium-containing stones (classes IIB and III) was found for the velocity of sound (p <0.01), the attenuation coefficient slope per time unit (p <0.10), the slope of the backscattering spectrum (p <0.05) and the 4.5 MHz intercept (p <0.01). B-mode classification yielded no complete distinction of these two groups of stones. It can be concluded that in vivo assessment of quantitative characteristics (front reflection, backscattering characteristics, attenuation coefficient slope per unit of time) in combination with the B-mode characteristics might be useful for in vivo gallstone classification.