The purpose of our study was to investigate the relationship between a twinkling artifact and the biochemical compositions of gallstones in phantom experiments. Fifty-two gallstones surgically extracted from 26 patients (26 cholesterol, 12 brown pigment and 14 black pigment stones) were included in the phantom study. Color Doppler sonography was performed to evaluate the occurrence of a twinkling artifact behind the stones. The artifacts were graded from grade 0 (no artifact) to grade 3 (very strong artifact). Micro computed tomography (CT) images were obtained to analyze the internal architecture and external surface of the stones. A total of 20 of 26 cholesterol stones (77%) produced grade 2 or 3 artifacts, whereas almost all the black pigment stones and more than half of the brown pigment stones produced no artifacts or grade 1 artifacts ( p = 0.0010). The sensitivity and specificity of grade 2 or greater artifacts for cholesterol stones were 76.9% and 76.9%, respectively. The composition of the stones was strongly correlated with the internal shape on the micro CT image. Cholesterol stones with rough surfaces did not show stronger intensities of the artifacts than those with smooth surfaces ( p > 0.05). In conclusion, most of the prominent artifacts arose from cholesterol stones having radial or radial-concentric internal architectures, whereas the black pigment stones mostly produced little artifact. (E-mail: leejy4u@snu.ac.kr)