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Previous studies have demonstrated that plaques from the internal carotid artery with lower median grayscale values are associated with higher complication rates in the perinterventional course. A repeatedly cited limitation of ultrasound is that the single plane used to calculate the median grayscale value is only two dimensional. The goal of this study was to compare the median grayscale value as determined using two dimensional cuts versus three dimensional data sets. Seventy-one cuts of 24 thromboendarterectomy samples from 19 patients were analyzed using 3D data sets. The ultrasound data sets were obtained using a 10 MHz probe in 3D mode in a special chamber and were evaluated by two investigators. Additionally, a longitudinal view of the samples was made using B mode according to a standardized protocol. Results: There was a significant correlation between the 2D and 3D data as assessed by two observers (p < 0.001, intraclass correlation [ICC] > 0.895) and at different times (p < 0,001, ICC > 0.935). Comparison of the 3D transverse cuts with the longitudinal B mode cuts also showed a highly significant association between the two methods (p < 0.001, R = 0.800). 97.2 % of the measured values were within the limits of agreement, reflecting the concordance of the both methods. The superiority of three dimensional ultrasound with respect to two dimensional ultrasound was not able to be demonstrated using this standardized in vitro procedure to examine the echolucency in extracranial internal carotid artery plaques. ; published